U.S. patent application number 14/762133 was filed with the patent office on 2015-11-05 for methods of saccharifying and fermenting a cellulosic material.
This patent application is currently assigned to Novozymes A/S. The applicant listed for this patent is NOVOZYMES A/S. Invention is credited to Jesper Frickmann, Armindo Ribiero Gaspar, Katja Salomon Johansen, Mark Stevens, Hui Xu.
Application Number | 20150315622 14/762133 |
Document ID | / |
Family ID | 51391846 |
Filed Date | 2015-11-05 |
United States Patent
Application |
20150315622 |
Kind Code |
A1 |
Frickmann; Jesper ; et
al. |
November 5, 2015 |
Methods of Saccharifying and Fermenting a Cellulosic Material
Abstract
The invention relates to methods of saccharifying a cellulosic
material comprising subjecting the cellulosic material to a
cellulolytic enzyme composition and a GH61 polypeptide, and
optionally a catalase in the presence of dissolved oxygen at a
concentration in the range of 0.5 to 10% of the saturation level.
The invention also related to methods of producing desired
fermentation products, such as ethanol, using a method including a
saccharification step of the invention.
Inventors: |
Frickmann; Jesper; (Raleigh,
NC) ; Gaspar; Armindo Ribiero; (Rolesville, NC)
; Stevens; Mark; (Kittrel, NC) ; Xu; Hui;
(Wake Forest, NC) ; Johansen; Katja Salomon;
(Gentofte, DK) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVOZYMES A/S |
Bagsvaerd |
|
DK |
|
|
Assignee: |
Novozymes A/S
Bagsvaerd
DK
|
Family ID: |
51391846 |
Appl. No.: |
14/762133 |
Filed: |
February 21, 2014 |
PCT Filed: |
February 21, 2014 |
PCT NO: |
PCT/US2014/017690 |
371 Date: |
July 20, 2015 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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61767488 |
Feb 21, 2013 |
|
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|
61898707 |
Nov 1, 2013 |
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Current U.S.
Class: |
435/99 ; 435/128;
435/136; 435/148; 435/155; 435/162 |
Current CPC
Class: |
C12P 5/023 20130101;
C12P 5/026 20130101; C12P 7/14 20130101; C12P 19/02 20130101; C12P
7/40 20130101; Y02E 50/10 20130101; C12P 7/10 20130101; C12Y 302/01
20130101; C12P 3/00 20130101; Y02E 50/17 20130101; C12P 19/14
20130101; Y02E 50/16 20130101; C12P 7/02 20130101; C12P 13/04
20130101 |
International
Class: |
C12P 19/14 20060101
C12P019/14; C12P 7/14 20060101 C12P007/14; C12P 19/02 20060101
C12P019/02 |
Claims
1. A method of saccharifying a cellulosic material comprising
subjecting the cellulosic material to a cellulolytic enzyme
composition and a GH61 polypeptide in a vessel, wherein oxygen is
added to the vessel to maintain a concentration of dissolved oxygen
in the range of 0.5 to 10% of the saturation level.
2. The method of claim 1, wherein the cellulosic material is
further subjected to a catalase.
3. The method of claim 1, wherein the amount of catalase is in the
range of 0.5% to 25%, e.g., 0.5% to 20%, 0.5% to 15%, 0.5% to 10%,
0.5% to 7.5%, 0.5% to 5%, and 0.5% to 4% of total protein.
4. The method of claim 1, wherein the dissolved oxygen
concentration during saccharification is in the range of 0.5-10% of
the saturation level, such as 0.5-7%, such as 0.5-5%, such as
0.5-4%, such as 0.5-3%, such as 0.5-2%, such as 1-5%, such as 1-4%,
such as 1-3%, such as 1-2% during at least 25%, such as at least
50%, such as at least 75% of the saccharification period.
5. The method of claim 1, further comprising adding a base to
maintain the pH in the range of about 3.0 to 7.0, e.g., 3.5 to 6.5,
4.0 to 6.0, 4.5 to 5.5 or about 5.0 during the
saccharification.
6. The method of claim 5, wherein the base is selected from the
group consisting of KOH, NaOH, Ca(OH).sub.2, and NH.sub.4OH.
7. The method of claim 1, wherein the cellulolytic enzyme
composition comprises a cellobiohydrolase, an endoglucanase, and a
beta-glucosidase.
8. The method of claim 1, wherein the cellulolytic enzyme
composition comprises a cellobiohydrolase I, a cellobiohydrolase
II, an endoglucanase, and a beta-glucosidase.
9. The method of claim 1, wherein the cellulolytic enzyme
composition comprises a cellobiohydrolase I, a cellobiohydrolase
II, an endoglucanase, a beta-glucosidase, and a xylanase.
10. The method of claim 1, wherein the cellulolytic enzyme
composition comprises a cellobiohydrolase I, a cellobiohydrolase
II, an endoglucanase, a beta-glucosidase, a xylanase, and a
beta-xylosidase.
11. The method of claim 1, wherein the vessel comprises more than
10 m.sup.3, such as more than 25 m.sup.3, such as more than 50
m.sup.3 cellulosic material.
12. A method of producing a fermentation product from cellulosic
material, comprising: (a) saccharification of the cellulosic
material in accordance with the method of claim 1; and (b)
fermenting the saccharified cellulosic material with one or more
fermenting microorganisms.
13. The method of claim 12, further comprising recovering the
fermentation product from (b).
14. The method of claim 12, wherein the fermentation product is an
alcohol, an organic acid, a ketone, an amino acid, or a gas.
15. The method of claim 14, wherein the fermentation product is
ethanol.
Description
REFERENCE TO A SEQUENCE LISTING
[0001] This application contains a Sequence Listing in computer
readable form, which is incorporated herein by reference.
BACKGROUND
[0002] Cellulosic material provides an attractive platform for
generating alternative energy sources to fossil fuels. The
conversion of cellulosic material (e.g., from lignocellulosic
feedstock) into biofuels has the advantages of the ready
availability of large amounts of feedstock, the desirability of
avoiding burning or land filling the materials, and the cleanliness
of the biofuels (such as ethanol). Wood, agricultural residues,
herbaceous crops, and municipal solid wastes have been considered
as feedstocks for biofuel production. Once the cellulosic material
is saccharified and converted to fermentable sugars, e.g., glucose,
the fermentable sugars may be fermented by yeast into biofuel, such
as ethanol.
[0003] New and improved enzymes and enzyme compositions have been
developed over the past decade and made saccharification of
pretreated cellulosic material more efficient. However, there is
still a need for improving saccharification of pretreated
cellulosic material and processes for producing biofuels.
SUMMARY OF THE INVENTION
[0004] Described herein are methods of saccharifying a cellulosic
material into fermentable sugars. Also described are methods of
producing fermentation products, such as ethanol, from a cellulosic
material, such as a pretreated cellulosic material, by
saccharification and fermentation.
[0005] In one aspect the invention relates to methods of
saccharifying a cellulosic material comprising subjecting the
cellulosic material to a cellulolytic enzyme composition and a GH61
polypeptide in the presence of dissolved oxygen at a concentration
in the range of 0.5 to 10% of the saturation level.
[0006] In another aspect the invention relates to methods of
producing a fermentation product, comprising:
[0007] (a) subjecting a cellulosic material to a cellulolytic
enzyme composition and a GH61 polypeptide in the presence of
dissolved oxygen at a concentration in the range of 0.5-10% of the
saturation level; [0008] (b) fermenting the saccharified cellulosic
material with one or more fermenting microorganisms; and
[0009] (c) recovering the fermentation product from (b).
[0010] In another aspect the invention relates methods of producing
a fermentation product, comprising:
[0011] (a) subjecting a cellulosic material to a cellulolytic
enzyme composition, a GH61 polypeptide, and a catalase in the
presence of dissolved oxygen at a concentration in the range of
0.5-10% of the saturation level;
[0012] (b) fermenting the saccharified cellulosic material with one
or more fermenting microorganisms; and
[0013] (c) recovering the fermentation product from (b).
[0014] In an embodiment the cellulosic material has been pretreated
e.g., by chemical and/or physical pretreatment, such as dilute acid
and/or steam explosion pretreatment. In a preferred embodiment the
cellulosic material is unwashed, such as unwashed pretreated corn
stover (uwPCS).
[0015] Methods of the present invention are used to
saccharify/hydrolyze a pretreated cellulosic material to sugars.
These sugars may be converted to many useful desired substances,
e.g., fuel, potable ethanol, and/or fermentation products (e.g.,
acids, alcohols, ketones, gases, and the like).
[0016] The saccharified pretreated cellulosic material may be
sugars that can be used in processes for producing syrups (e.g.,
High Fructose Corn Syrups (HFCS) and/or plastics (e.g.,
polyethylene, polystyrene, and polypropylene), polylactic acid
(e.g., for producing PET).
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 shows the effect of dissolved oxygen (DO) on glucose
yield (g/L) after 5 days saccharification using a cellulolytic
enzyme composition with and without Thermoascus auranticus GH61A
polypeptide (GH61 Polypeptide A).
[0018] FIG. 2 shows the effect of dissolved oxygen (DO) on glucose
yield (g/L) after 5 days saccharification using a cellulolytic
enzyme composition with Penicillium emersonii GH61 polypeptide
(GH61 Polypeptide B).
DEFINITIONS
[0019] Alpha-L-arabinofuranosidase: The term
"alpha-L-arabinofuranosidase" means an alpha-L-arabinofuranoside
arabinofuranohydrolase (EC 3.2.1.55) that catalyzes the hydrolysis
of terminal non-reducing alpha-L-arabinofuranoside residues in
alpha-L-arabinosides. The enzyme acts on
alpha-L-arabinofuranosides, alpha-L-arabinans containing (1,3)-
and/or (1,5)-linkages, arabinoxylans, and arabinogalactans.
Alpha-L-arabinofuranosidase is also known as arabinosidase,
alpha-arabinosidase, alpha-L-arabinosidase,
alpha-arabinofuranosidase, polysaccharide
alpha-L-arabinofuranosidase, alpha-L-arabinofuranoside hydrolase,
L-arabinosidase, or alpha-L-arabinanase. For purposes of the
present invention, alpha-L-arabinofuranosidase activity is
determined using 5 mg of medium viscosity wheat arabinoxylan
(Megazyme International Ireland, Ltd., Bray, Co. Wicklow, Ireland)
per ml of 100 mM sodium acetate pH 5 in a total volume of 200
microliters for 30 minutes at 40.degree. C. followed by arabinose
analysis by AMINEX.RTM. HPX-87H column chromatography (Bio-Rad
Laboratories, Inc., Hercules, Calif., USA).
[0020] Alpha-glucuronidase: The term "alpha-glucuronidase" means an
alpha-D-glucosiduronate glucuronohydrolase (EC 3.2.1.139) that
catalyzes the hydrolysis of an alpha-D-glucuronoside to
D-glucuronate and an alcohol. For purposes of the present
invention, alpha-glucuronidase activity is determined according to
de Vries, 1998, J. Bacteriol. 180: 243-249. One unit of
alpha-glucuronidase equals the amount of enzyme capable of
releasing 1 micromole of glucuronic or 4-O-methylglucuronic acid
per minute at pH 5, 40.degree. C.
[0021] Beta-glucosidase: The term "beta-glucosidase" means a
beta-D-glucoside glucohydrolase (E.C. 3.2.1.21), which catalyzes
the hydrolysis of terminal non-reducing beta-D-glucose residues
with the release of beta-D-glucose. For purposes of the present
invention, beta-glucosidase activity is determined according to the
basic procedure described by Venturi et al., 2002, Extracellular
beta-D-glucosidase from Chaetomium thermophilum var. coprophilum:
production, purification and some biochemical properties, J. Basic
Microbiol. 42: 55-66. One unit of beta-glucosidase is defined as
1.0 micromole of p-nitrophenolate anion produced per minute at
25.degree. C., pH 4.8 from 1 mM
p-nitrophenyl-beta-D-glucopyranoside as substrate in 50 mM sodium
citrate containing 0.01% TWEEN.RTM. 20.
[0022] Beta-xylosidase: The term "beta-xylosidase" means a
beta-D-xyloside xylohydrolase (E.C. 3.2.1.37) that catalyzes the
exo-hydrolysis of short beta (1.fwdarw.4)-xylooligosaccharides, to
remove successive D-xylose residues from the non-reducing termini.
Beta-xylosidase activity can be determined using 1 mM
p-nitrophenyl-beta-D-xyloside as substrate in 100 mM sodium citrate
containing 0.01% TWEEN.RTM. 20 at pH 5, 40.degree. C. For purposes
of the present invention, one unit of beta-xylosidase is defined as
1.0 micromole of p-nitrophenolate anion produced per minute at
40.degree. C., pH 5 from 1 mM p-nitrophenyl-beta-D-xyloside as
substrate in 100 mM sodium citrate containing 0.01% TWEEN.RTM.
20.
[0023] Biomass material: The term "biomass material" refers to any
sugar-containing biomass (e.g., stems, leaves, hulls, husks, and
cobs of plants or leaves, branches, and wood of trees) and any
component thereof, such as cellulose, hemicellulose, or lignan. It
is understood that, unless otherwise specified, biomass material
includes untreated, pretreated, and hydrolyzed or partially
hydrolyzed forms (e.g., biomass degraded products, such as
oligosaccharides).
[0024] Catalase: The term "catalase" means a hydrogen-peroxide:
hydrogen-peroxide oxidoreductase (E.C. 1.11.1.6 or E.C. 1.11.1.21)
that catalyzes the conversion of two hydrogen peroxides to oxygen
and two waters. Catalase activity can be determined by monitoring
the degradation of hydrogen peroxide at 240 nm based on the
following reaction:
2H.sub.2O.sub.2.fwdarw.2H.sub.2O+O.sub.2
[0025] The reaction is conducted in 50 mM phosphate pH 7 at
25.degree. C. with 10.3 mM substrate (H.sub.2O.sub.2) and
approximately 100 units of enzyme per ml. Absorbance is monitored
spectrophotometrically within 16-24 seconds, which should
correspond to an absorbance reduction from 0.45 to 0.4. One
catalase activity unit can be expressed as one micromole of
H.sub.2O.sub.2 degraded per minute at pH 7.0 and 25.degree. C.
[0026] cDNA: The term "cDNA" means a DNA molecule that can be
prepared by reverse transcription from a mature, spliced, mRNA
molecule obtained from a eukaryotic or prokaryotic cell. cDNA lacks
intron sequences that may be present in the corresponding genomic
DNA. The initial, primary RNA transcript is a precursor to mRNA
that is processed through a series of steps, including splicing,
before appearing as mature spliced mRNA.
[0027] Cellobiohydrolase: The term "cellobiohydrolase" means a
1,4-beta-D-glucan cellobiohydrolase (E.C. 3.2.1.91) that catalyzes
the hydrolysis of 1,4-beta-D-glucosidic linkages in cellulose,
cellooligosaccharides, or any beta-1,4-linked glucose containing
polymer, releasing cellobiose from the reducing or non-reducing
ends of the chain (Teeri, 1997, Crystalline cellulose degradation:
New insight into the function of cellobiohydrolases, Trends in
Biotechnology 15: 160-167; Teen et al., 1998, Trichoderma reesei
cellobiohydrolases: why so efficient on crystalline cellulose?,
Biochem. Soc. Trans. 26: 173-178). Cellobiohydrolase activity is
determined according to the procedures described by Lever et al.,
1972, Anal. Biochem. 47: 273-279; van Tilbeurgh et al., 1982, FEBS
Letters, 149: 152-156; van Tilbeurgh and Claeyssens, 1985, FEBS
Letters, 187: 283-288; and Tomme et al., 1988, Eur. J. Biochem.
170: 575-581. In the present invention, the Tomme et al. method can
be used to determine cellobiohydrolase activity.
[0028] Cellulolytic enzyme composition: The term "cellulolytic
enzyme composition" means one or more (e.g., several) enzymes that
hydrolyze a cellulosic material. Such enzymes include
endoglucanase(s), cellobiohydrolase(s), beta-glucosidase(s), or
combinations thereof. The two basic approaches for measuring
cellulolytic activity include: (1) measuring the total cellulolytic
activity, and (2) measuring the individual cellulolytic activities
(endoglucanases, cellobiohydrolases, and beta-glucosidases) as
reviewed in Zhang et al., 2006, Outlook for cellulase improvement:
Screening and selection strategies, Biotechnology Advances 24:
452-481. Total cellulolytic activity is usually measured using
insoluble substrates, including Whatman No 1 filter paper,
microcrystalline cellulose, bacterial cellulose, algal cellulose,
cotton, pretreated lignocellulose, etc. The most common total
cellulolytic activity assay is the filter paper assay using Whatman
No 1 filter paper as the substrate. The assay was established by
the International Union of Pure and Applied Chemistry (IUPAC)
(Ghose, 1987, Measurement of cellulase activities, Pure Appl. Chem.
59: 257-68).
[0029] For purposes of the present invention, cellulolytic enzyme
activity is determined by measuring the increase in
hydrolysis/saccharification of a cellulosic material by
cellulolytic enzyme(s) under the following conditions: 1-50 mg of
cellulolytic enzyme protein/g of cellulose in PCS (or other
pretreated cellulosic material) for 3-7 days at a suitable
temperature, e.g., 50.degree. C., 55.degree. C., 60.degree. C., or
65.degree. C., compared to a control hydrolysis without addition of
cellulolytic enzyme protein. Typical conditions are 1 ml reactions,
washed or unwashed PCS, 5% insoluble solids, 50 mM sodium acetate
pH 5, 1 mM MnSO.sub.4, 50.degree. C., 55.degree. C., 60.degree. C.,
or 65.degree. C., 72 hours, sugar analysis by AMINEX.RTM. HPX-87H
column (Bio-Rad Laboratories, Inc., Hercules, Calif., USA).
[0030] Cellulosic material: The term "cellulosic material" refers
to any biomass material containing cellulose (a chemically
homogeneous oligosaccharide or polysaccharide of
beta-(1-4)-D-glucan (polymer containing beta (1-4) linked D-glucose
units)). Although generally polymorphous, cellulose can be found in
plant tissue primarily as an insoluble crystalline matrix of
parallel glucan chains. Cellulose is generally found, for example,
in the stems, leaves, hulls, husks, and cobs of plants or leaves,
branches, and wood of trees. The cellulosic material can be, but is
not limited to, herbaceous material, agricultural residue, forestry
residue, municipal solid waste, waste paper, and pulp and paper
mill residue (see, for example, Wiselogel et al., 1995, in Handbook
on Bioethanol (Charles E. Wyman, editor), pp. 105-118, Taylor &
Francis, Washington D.C.; Wyman, 1994, Bioresource Technology 50:
3-16; Lynd, 1990, Applied Biochemistry and Biotechnology 24/25:
695-719; Mosier et al., 1999, Recent Progress in Bioconversion of
Lignocellulosics, in Advances in Biochemical
Engineering/Biotechnology, T. Scheper, managing editor, Volume 65,
pp. 23-40, Springer-Verlag, New York). Cellulosic material includes
any form of cellulose, such as polysaccharides degraded or
hydrolyzed to oligosaccharides. It is understood herein that the
cellulose may be in the form of a component of lignocellulose, a
plant cell wall material containing lignin, cellulose, and
hemicellulose in a mixed matrix.
[0031] In one aspect, the cellulosic material is herbaceous
material (including energy crops). In another aspect, the
cellulosic material is agricultural residue. In another aspect, the
cellulosic material is wood (including forestry residue). In
another aspect, the cellulosic material is municipal solid waste.
In another aspect, the cellulosic material is waste paper. In
another aspect, the cellulosic material is pulp and paper mill
residue.
[0032] In another aspect, the cellulosic material is corn stover.
In another aspect, the cellulosic material is wheat straw. In
another aspect, the cellulosic material is bagasse. In another
aspect, the cellulosic material is corn cob. In another aspect, the
cellulosic material is switchgrass. In another aspect, the
cellulosic material is corn fiber. In another aspect, the
cellulosic material is rice straw. In another aspect, the
cellulosic material is miscanthus. In another aspect, the
cellulosic material is arundo. In another aspect, the cellulosic
material is bamboo. In another aspect, the cellulosic material is
orange peel. In another aspect, the cellulosic material is poplar.
In another aspect, the cellulosic material is pine. In another
aspect, the cellulosic material is aspen. In another aspect, the
cellulosic material is fir. In another aspect, the cellulosic
material is spuce. In another aspect, the cellulosic material is
willow. In another aspect, the cellulosic material is
eucalyptus.
[0033] In another aspect, the cellulosic material is
microcrystalline cellulose. In another aspect, the cellulosic
material is bacterial cellulose. In another aspect, the cellulosic
material is algal cellulose. In another aspect, the cellulosic
material is cotton linter. In another aspect, the cellulosic
material is amorphous phosphoric-acid treated cellulose. In another
aspect, the cellulosic material is filter paper.
[0034] In another aspect, the cellulosic material is an aquatic
biomass. As used herein the term "aquatic biomass" means biomass
produced in an aquatic environment by a photosynthesis process. The
aquatic biomass can be algae; submerged plants; emergent plants;
and floating-leaf plants.
[0035] The cellulosic material may be used as is or may be
subjected to pretreatment (pretreated cellulosic material), using
conventional methods known in the art, as described herein.
[0036] Coding sequence: The term "coding sequence" means a
polynucleotide, which directly specifies the amino acid sequence of
a polypeptide. The boundaries of the coding sequence are generally
determined by an open reading frame, which usually begins with the
ATG start codon or alternative start codons such as GTG and TTG and
ends with a stop codon such as TAA, TAG, and TGA. The coding
sequence may be a DNA, cDNA, synthetic, or recombinant
polynucleotide.
[0037] Dissolved Oxygen Saturation Level: The saturation level of
oxygen is determined at the standard partial pressure (0.21
atmosphere) of oxygen. The saturation level at the standard partial
pressure of oxygen is dependent on the temperature and solute
concentrations. In an embodiment where the temperature during
hydrolysis is 50.degree. C., the saturation level would typically
be in the range of 5-5.5 mg oxygen per kg slurry, depending on the
solute concentrations. Hence, dissolved oxygen is present in a
range from 0.025 ppm to 0.55 ppm, such as, e.g., 0.05 to 0.165 ppm
at temperatures around 50.degree. C.
[0038] Endoglucanase: The term "endoglucanase" means an
endo-1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase (E.C. 3.2.1.4),
which catalyzes endohydrolysis of 1,4-beta-D-glycosidic linkages in
cellulose, cellulose derivatives (such as carboxymethyl cellulose
and hydroxyethyl cellulose), lichenin, beta-1,4 bonds in mixed
beta-1,3 glucans such as cereal beta-D-glucans or xyloglucans, and
other plant material containing cellulosic components.
Endoglucanase activity can be determined by measuring reduction in
substrate viscosity or increase in reducing ends determined by a
reducing sugar assay (Zhang et al., 2006, Biotechnology Advances
24: 452-481). For purposes of the present invention, endoglucanase
activity is determined using carboxymethyl cellulose (CMC) as
substrate according to the procedure of Ghose, 1987, Pure and Appl.
Chem. 59: 257-268, at pH 5, 40.degree. C.
[0039] Family 61 glycoside hydrolase: The term "Family 61 glycoside
hydrolase" or "Family GH61" or "GH61 polypeptide" means a
polypeptide falling into the glycoside hydrolase Family 61
according to Henrissat, 1991, A classification of glycosyl
hydrolases based on amino-acid sequence similarities, Biochem. J.
280: 309-316, and Henrissat and Bairoch, 1996, Updating the
sequence-based classification of glycosyl hydrolases, Biochem. J.
316: 695-696. The enzymes in this family were originally classified
as a glycoside hydrolase family based on measurement of very weak
endo-1,4-beta-D-glucanase activity in one family member. GH61
polypeptides are now classified as lytic polysaccharide
monooxygenases (Quinlan et al., 2011, Proc. Natl. Acad. Sci. USA
208: 15079-15084; Phillips et al., 2011, ACS Chem. Biol. 6:
1399-1406; Lin et al., 2012, Structure 20: 1051-1061) and placed
into a new family designated "Auxiliary Activity 9" or "AA9".
[0040] GH61 polypeptides enhance hydrolysis/saccharification of a
cellulosic material by an enzyme having cellulolytic activity. For
purposes of the present invention, cellulolytic enhancing activity
is determined by measuring the increase in reducing sugars or the
increase of the total of cellobiose and glucose from the hydrolysis
of a cellulosic material by cellulolytic enzyme under the following
conditions: 1-50 mg of total protein/g of cellulose in PCS, wherein
total protein is comprised of 50-99.5% w/w cellulolytic enzyme
protein and 0.5-50% w/w protein of a GH61 polypeptide for 1-7 days
at a suitable temperature, e.g., 50.degree. C., 55.degree. C., or
60.degree. C., compared to a control hydrolysis with equal total
protein loading without cellulolytic enhancing activity (1-50 mg of
cellulolytic protein/g of cellulose in PCS). In a preferred aspect,
a mixture of CELLUCLAST.RTM. 1.5L (Novozymes A/S, Bagsvrd, Denmark)
in the presence of 2-3% of total protein weight Aspergillus oryzae
beta-glucosidase (recombinantly produced in Aspergillus oryzae
according to WO 02/095014) or 2-3% of total protein weight
Aspergillus fumigatus beta-glucosidase (recombinantly produced in
Aspergillus oryzae as described in WO 02/095014) of cellulase
protein loading is used as the source of the cellulolytic
activity.
[0041] GH61 polypeptides enhance the hydrolysis/saccharification of
a cellulosic material catalyzed by enzyme having cellulolytic
activity by reducing the amount of cellulolytic enzyme required to
reach the same degree of hydrolysis preferably at least 1.01-fold,
more preferably at least 1.05-fold, more preferably at least
1.10-fold, more preferably at least 1.25-fold, more preferably at
least 1.5-fold, more preferably at least 2-fold, more preferably at
least 3-fold, more preferably at least 4-fold, more preferably at
least 5-fold, even more preferably at least 10-fold, and most
preferably at least 20-fold.
[0042] Feruloyl esterase: The term "feruloyl esterase" means a
4-hydroxy-3-methoxycinnamoyl-sugar hydrolase (EC 3.1.1.73) that
catalyzes the hydrolysis of the 4-hydroxy-3-methoxycinnamoyl
(feruloyl) group from an esterified sugar, which is usually
arabinose in "natural" substrates, to produce ferulate
(4-hydroxy-3-methoxycinnamate). Feruloyl esterase is also known as
ferulic acid esterase, hydroxycinnamoyl esterase, FAE-III,
cinnamoyl ester hydrolase, FAEA, cinnAE, FAE-I, or FAE-II. For
purposes of the present invention, feruloyl esterase activity is
determined using 0.5 mM p-nitrophenylferulate as substrate in 50 mM
sodium acetate pH 5.0. One unit of feruloyl esterase equals the
amount of enzyme capable of releasing 1 micromole of
p-nitrophenolate anion per minute at pH 5, 25.degree. C.
[0043] Hemicellulose: As used herein, the term "hemicellulose"
refers to an oligosaccharide or polysaccharide of biomass material
other than cellulose. Hemicellulose is chemically heterogeneous and
includes a variety of polymerized sugars, primarily D-pentose
sugars, such as xylans, xyloglucans, arabinoxylans, and mannans, in
complex heterogeneous branched and linear polysaccharides or
oligosaccharides that are bound via hydrogen bonds to the cellulose
microfibrils in the plant cell wall, and wherein xylose sugars are
usually in the largest amount. Hemicelluloses may be covalently
attached to lignin, and usually hydrogen bonded to cellulose, as
well as to other hemicelluloses, which help stabilize the cell wall
matrix forming a highly complex structure. Hemicellulosic material
includes any form of hemicellulose, such as polysaccharides
degraded or hydrolyzed to oligosaccharides. It is understood herein
that the hemicellulose may be in the form of a component of
lignocellulose, a plant cell wall material containing lignin,
cellulose, and hemicellulose in a mixed matrix.
[0044] Hemicellulolytic enzyme or hemicellulase: The term
"hemicellulolytic enzyme" or "hemicellulase" means one or more
(several) enzymes that hydrolyze a hemicellulosic material. See,
for example, Shallom and Shoham, 2003, Microbial hemicellulases,
Current Opinion In Microbiology 6(3): 219-228). Hemicellulases are
key components in the degradation of plant biomass. Examples of
hemicellulases include, but are not limited to, an acetylmannan
esterase, an acetyxylan esterase, an arabinanase, an
arabinofuranosidase, a coumaric acid esterase, a feruloyl esterase,
a galactosidase, a glucuronidase, a glucuronoyl esterase, a
mannanase, a mannosidase, a xylanase, and a xylosidase. The
catalytic modules of hemicellulases are either glycoside hydrolases
(GHs) that hydrolyze glycosidic bonds, or carbohydrate esterases
(CEs), which hydrolyze ester linkages of acetate or ferulic acid
side groups. These catalytic modules, based on homology of their
primary sequence, can be assigned into GH and CE families marked by
numbers. Some families, with overall similar fold, can be further
grouped into clans, marked alphabetically (e.g., GH-A). A most
informative and updated classification of these and other
carbohydrate active enzymes is available on the Carbohydrate-Active
Enzymes (CAZy) database. Hemicellulolytic enzyme activities can be
measured according to Ghose and Bisaria, 1987, Pure & Appl.
Chem. 59: 1739-1752.
[0045] Host cell: The term "host cell" means any cell type that is
susceptible to transformation, transfection, transduction, or the
like with a nucleic acid construct or expression vector comprising
a polynucleotide. The term "host cell" encompasses any progeny of a
parent cell that is not identical to the parent cell due to
mutations that occur during replication.
[0046] Isolated: The term "isolated" means a substance in a form or
environment that does not occur in nature. Non-limiting examples of
isolated substances include (1) any non-naturally occurring
substance, (2) any substance including, but not limited to, any
enzyme, variant, nucleic acid, protein, peptide or cofactor, that
is at least partially removed from one or more or all of the
naturally occurring constituents with which it is associated in
nature; (3) any substance modified by the hand of man relative to
that substance found in nature; or (4) any substance modified by
increasing the amount of the substance relative to other components
with which it is naturally associated (e.g., multiple copies of a
gene encoding the substance, or use of a stronger promoter than the
promoter naturally associated with the gene encoding the
substance). An isolated substance may be present in a fermentation
broth.
[0047] Mature polypeptide: The term "mature polypeptide" means a
polypeptide in its final form following translation and any
post-translational modifications, such as N-terminal processing,
C-terminal truncation, glycosylation, phosphorylation, etc. It is
known in the art that a host cell may produce a mixture of two of
more different mature polypeptides (i.e., with a different
C-terminal and/or N-terminal amino acid) expressed by the same
polynucleotide. The mature polypeptide can be predicted using the
SignalP program (Nielsen et al., 1997, Protein Engineering 10:
1-6).
[0048] Mature polypeptide coding sequence: The term "mature
polypeptide coding sequence" is defined herein as a nucleotide
sequence that encodes a mature polypeptide having biological
activity. The mature polypeptide coding sequence can be predicted
using the SignalP program (Nielsen et al., 1997, supra).
[0049] Nucleic acid construct: The term "nucleic acid construct"
means a nucleic acid molecule, either single- or double-stranded,
which is isolated from a naturally occurring gene or is modified to
contain segments of nucleic acids in a manner that would not
otherwise exist in nature or which is synthetic, which comprises
one or more control sequences.
[0050] Polypeptide fragment: The term "fragment" means a
polypeptide having one or more (e.g., several) amino acids deleted
from the amino and/or carboxyl terminus of a mature polypeptide. In
one aspect, a fragment contains at least 85% of the amino acid
residues, e.g., at least 90% of the amino acid residues or at least
95% of the amino acid residues of the referenced mature
polypeptide.
[0051] Stringency conditions: For long probes of at least 100
nucleotides in length, very low to very high stringency conditions
are defined as prehybridization and hybridization at 42.degree. C.
in 5.times.SSPE, 0.3% SDS, 200 micrograms/ml sheared and denatured
salmon sperm DNA, and either 25% formamide for very low and low
stringencies, 35% formamide for medium and medium-high
stringencies, or 50% formamide for high and very high stringencies,
following standard Southern blotting procedures for 12 to 24 hours
optimally. The carrier material is finally washed three times each
for 15 minutes using 2.times.SSC, 0.2% SDS at 45.degree. C. (very
low stringency), at 50.degree. C. (low stringency), at 55.degree.
C. (medium stringency), at 60.degree. C. (medium-high stringency),
at 65.degree. C. (high stringency), and at 70.degree. C. (very high
stringency).
[0052] For short probes of about 15 nucleotides to about 70
nucleotides in length, stringency conditions are defined as
prehybridization and hybridization at about 5.degree. C. to about
10.degree. C. below the calculated Tm using the calculation
according to Bolton and McCarthy (1962, Proc. Natl. Acad. Sci. USA
48: 1390) in 0.9 M NaCl, 0.09 M Tris HCl pH 7.6, 6 mM EDTA, 0.5% NP
40, 1.times.Denhardt's solution, 1 mM sodium pyrophosphate, 1 mM
sodium monobasic phosphate, 0.1 mM ATP, and 0.2 mg of yeast RNA per
ml following standard Southern blotting procedures for 12 to 24
hours optimally. The carrier material is finally washed once in
6.times.SCC plus 0.1% SDS for 15 minutes and twice each for 15
minutes using 6.times.SSC at 5.degree. C. to 10.degree. C. below
the calculated Tm.
[0053] Parent Enzyme: The term "parent" means an enzyme to which an
alteration is made to produce a variant. The parent may be a
naturally occurring (wild-type) polypeptide or a variant
thereof.
[0054] Pretreated corn stover: The term "PCS" or "Pretreated Corn
Stover" means a cellulosic material derived from corn stover by
pretreatment (e.g., by heat and dilute sulfuric acid, alkaline
pretreatment, or neutral pretreatment).
[0055] Sequence identity: The relatedness between two amino acid
sequences or between two nucleotide sequences is described by the
parameter "sequence identity".
[0056] For purposes of the present invention, the sequence identity
between two amino acid sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, J. Mol.
Biol. 48: 443-453) as implemented in the Needle program of the
EMBOSS package (EMBOSS: The European Molecular Biology Open
Software Suite, Rice et al., 2000, Trends Genet. 16: 276-277),
preferably version 5.0.0 or later. The parameters used are gap open
penalty of 10, gap extension penalty of 0.5, and the EBLOSUM62
(EMBOSS version of BLOSUM62) substitution matrix. The output of
Needle labeled "longest identity" (obtained using the -nobrief
option) is used as the percent identity and is calculated as
follows:
(Identical Residues.times.100)/(Length of Alignment-Total Number of
Gaps in Alignment)
[0057] For purposes of the present invention, the sequence identity
between two deoxyribonucleotide sequences is determined using the
Needleman-Wunsch algorithm (Needleman and Wunsch, 1970, supra) as
implemented in the Needle program of the EMBOSS package (EMBOSS:
The European Molecular Biology Open Software Suite, Rice et al.,
2000, supra), preferably version 5.0.0 or later. The parameters
used are gap open penalty of 10, gap extension penalty of 0.5, and
the EDNAFULL (EMBOSS version of NCBI NUC4.4) substitution matrix.
The output of Needle labeled "longest identity" (obtained using the
-nobrief option) is used as the percent identity and is calculated
as follows:
(Identical Deoxyribonucleotides.times.100)/(Length of
Alignment-Total Number of Gaps in Alignment)
[0058] Subsequence: The term "subsequence" means a polynucleotide
having one or more (several) nucleotides deleted from the 5' and/or
3' end of a mature polypeptide coding sequence; wherein the
subsequence encodes a fragment having biological activity.
[0059] Variant: The term "variant" means a chitin binding protein
comprising an alteration, i.e., a substitution, insertion, and/or
deletion of one or more (e.g., several) amino acid residues at one
or more positions. A substitution means a replacement of the amino
acid occupying a position with a different amino acid; a deletion
means removal of the amino acid occupying a position; and an
insertion means adding an amino acid adjacent to the amino acid
occupying a position.
[0060] Xylan degrading activity or xylanolytic activity: The term
"xylan degrading activity" or "xylanolytic activity" means a
biological activity that hydrolyzes xylan-containing material. The
two basic approaches for measuring xylanolytic activity include:
(1) measuring the total xylanolytic activity, and (2) measuring the
individual xylanolytic activities (e.g., endoxylanases,
beta-xylosidases, arabinofuranosidases, alpha-glucuronidases,
acetylxylan esterases, feruloyl esterases, and alpha-glucuronyl
esterases). Recent progress in assays of xylanolytic enzymes was
summarized in several publications including Biely and Puchard,
Recent progress in the assays of xylanolytic enzymes, 2006, Journal
of the Science of Food and Agriculture 86(11): 1636-1647; Spanikova
and Biely, 2006, Glucuronoyl esterase--Novel carbohydrate esterase
produced by Schizophyllum commune, FEBS Letters 580(19): 4597-4601;
Herrmann et al., 1997, The beta-D-xylosidase of Trichoderma reesei
is a multifunctional beta-D-xylan xylohydrolase, Biochemical
Journal 321: 375-381.
[0061] Total xylan degrading activity can be measured by
determining the reducing sugars formed from various types of xylan,
including, for example, oat spelt, beechwood, and larchwood xylans,
or by photometric determination of dyed xylan fragments released
from various covalently dyed xylans. The most common total
xylanolytic activity assay is based on production of reducing
sugars from polymeric 4-O-methyl glucuronoxylan as described in
Bailey et al., 1992, Interlaboratory testing of methods for assay
of xylanase activity, Journal of Biotechnology 23(3): 257-270.
Xylanase activity can also be determined with 0.2%
AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100 and 200
mM sodium phosphate buffer pH 6 at 37.degree. C. One unit of
xylanase activity is defined as 1.0 micromole of azurine produced
per minute at 37.degree. C., pH 6 from 0.2% AZCL-arabinoxylan as
substrate in 200 mM sodium phosphate pH 6 buffer.
[0062] For purposes of the present invention, xylan degrading
activity is determined by measuring the increase in hydrolysis of
birchwood xylan (Sigma Chemical Co., Inc., St. Louis, Mo., USA) by
xylan-degrading enzyme(s) under the following typical conditions: 1
ml reactions, 5 mg/ml substrate (total solids), 5 mg of xylanolytic
protein/g of substrate, 50 mM sodium acetate pH 5, 50.degree. C.,
24 hours, sugar analysis using p-hydroxybenzoic acid hydrazide
(PHBAH) assay as described by Lever, 1972, A new reaction for
colorimetric determination of carbohydrates, Anal. Biochem. 47:
273-279.
[0063] Xylanase: The term "xylanase" means a
1,4-beta-D-xylan-xylohydrolase (E.C. 3.2.1.8) that catalyzes the
endohydrolysis of 1,4-beta-D-xylosidic linkages in xylans. For
purposes of the present invention, xylanase activity is determined
with 0.2% AZCL-arabinoxylan as substrate in 0.01% TRITON.RTM. X-100
and 200 mM sodium phosphate buffer pH 6 at 37.degree. C. One unit
of xylanase activity is defined as 1.0 micromole of azurine
produced per minute at 37.degree. C., pH 6 from 0.2%
AZCL-arabinoxylan as substrate in 200 mM sodium phosphate pH 6
buffer.
[0064] Reference to "about" a value or parameter herein includes
aspects that are directed to that value or parameter per se. For
example, description referring to "about X" includes the aspect
"X".
[0065] As used herein and in the appended claims, the singular
forms "a," "or," and "the" include plural referents unless the
context clearly dictates otherwise. It is understood that the
aspects of the invention described herein include "consisting"
and/or "consisting essentially of" aspects.
[0066] Unless defined otherwise or clearly indicated by context,
all technical and scientific terms used herein have the same
meaning as commonly understood by one of ordinary skill in the art
to which this invention belongs.
DETAILED DESCRIPTION
[0067] The present invention relates to, inter alia, methods of
saccharifying a cellulosic material into sugars, such as
fermentable sugars, and converting these sugars into desired
products.
[0068] The fermentable sugars may be converted to many useful
desired substances, e.g., fuel, potable ethanol, and/or
fermentation products (e.g., acids, alcohols, ketones, gases, and
the like).
[0069] The saccharified pretreated cellulosic material may also be
sugars that can be used in processes for producing syrups (e.g.,
High Fructose Corn Syrups (HFCS) and/or plastics (e.g.,
polyethylene, polystyrene, and polypropylene), polylactic acid
(e.g., for producing PET).
[0070] The inventors have surprisingly found that the presence of
dissolved oxygen (DO) in the range of 0.5-10% of the saturation
level during enzymatic saccharification gives a strong increase in
glucose yield, when using a cellulolytic enzyme composition and a
GH61 polypeptide. The inventors found that for cellulolytic enzyme
compositions without a GH61 polypeptide, dissolved oxygen (DO) has
little or no effect on the fermentable sugar yield (see Example 1).
However, for cellulolytic enzyme compositions with a GH61
polypeptide, the dissolved oxygen (DO) concentration has a strong
effect on the final yield of fermentable sugars, with an optimum DO
concentration in the range of around 1-2% of the saturation level
(See Examples 1 and 2).
Methods of Saccharifying Cellulosic Materials
[0071] In one aspect the invention relates to methods of
saccharifying a cellulosic material comprising subjecting the
cellulosic material to a cellulolytic enzyme composition and a GH61
polypeptide in the presence of dissolved oxygen at a concentration
in the range of 0.5 to 10% of the dissolved oxygen saturation
level.
[0072] In another aspect the invention relates to methods of
saccharifying a cellulosic material comprising subjecting the
cellulosic material to a cellulolytic enzyme composition, a GH61
polypeptide and a catalase in the presence of dissolved oxygen at a
concentration in the range of 0.5 to 10% of the saturation
level.
[0073] In the saccharification step, also known as hydrolysis, the
cellulosic material, e.g., pretreated cellulosic material, is
treated to break down cellulose and/or hemicellulose to fermentable
sugars, such as arabinose, cellobiose, galactose, glucose, mannose,
xylose, xylulose, and/or soluble oligosaccharides. The
saccharification is performed enzymatically by a cellulolytic
enzyme composition and a GH61 polypeptide. The enzymes of the
compositions can be added simultaneously or sequentially. For
instance the GH61 polypeptide may be comprised in the cellulolytic
enzyme composition.
[0074] Enzymatic saccharification is preferably carried out in a
suitable aqueous environment under conditions that can be readily
determined by one skilled in the art and in the presence of
dissolved oxygen as defined herein. In one aspect, saccharification
is performed under conditions suitable for the activity of the
enzyme(s), i.e., optimal for the enzyme(s). The saccharification
may be carried out as a fed batch or continuous process where the,
e.g., pretreated, cellulosic material (substrate) is fed gradually
to, for example, an enzyme containing saccharification
solution.
[0075] According to the invention saccharification may
advantageously be performed in stirred-tank reactors, vessels,
tanks or fermentors under controlled pH, temperature, and oxygen,
and mixing conditions. In an embodiment, the reactor, vessel, tank
or fermentor comprises more than 10 m.sup.3, such as more than 25
m.sup.3, such as more than 50 m.sup.3 cellulosic material.
[0076] Saccharification may occur for up to 200 hours, e.g., about
12 to about 96 hours, about 16 to about 72 hours, or about 24 to
about 48 hours, such as for at least 12 hours, e.g., at least 24
hours, at least 36 hours, at least 48 hours, at least 60 hours, or
at least 72 hours.
[0077] In an embodiment saccharification is performed at a
temperature in the range of about 25.degree. C. to about 75.degree.
C., e.g., about 30.degree. C. to about 70.degree. C., about
35.degree. C. to about 65.degree. C., about 40.degree. C. to
60.degree. C., about 45.degree. C. to 55.degree. C., or about
50.degree. C.
[0078] In an embodiment saccharification is performed at a pH in
the range of about 3.0 to aout 7.0, e.g., 3.5 to 6.5, 4.0 to 6.0,
4.5 to 5.5 or about 5.0. In an embodiment, the process of the
present invention further comprises adding a base to the tank to
maintain the pH in the range of about 3.0 to about 7.0, e.g., 3.5
to 6.5, 4.0 to 6.0, 4.5 to 5.5 or about 5.0. Any base may be used,
including but not limited to KOH, NaOH, Ca(OH).sub.2, and
NH.sub.4OH or a combination thereof. In an embodiment, the base is
added in an amount of 25-2,500 mmol base per kg dry cellulosic
material, such as 25-1000 mmol/kg, 25-500 mmol/kg, 25-250 mmol/kg,
50-200 mmol/kg. The inventors have determined that less base is
required to maintain the pH during saccharification of a cellulosic
material with a cellulolytic composition and a GH61 polypeptide in
the presence of a catalase compared to the same process with the
cellulolytic composition and the GH61 polypeptide but in the
absence of a catalase. Thus, the processes of the present invention
involving a catalase are less costly and produce less waste in the
form of salt. In addition, the processes of the present invention
make it easier to recycle spent biomass to the fields, due to the
lower content of salts originating from the added base. In an
embodiment, the amount of base added during saccharification is
reduced by at least 1%, e.g., at least 2.5%, at least 5%, at least
10%, at least 15%, at least 20%, at least 25%, at least 30%, at
least 35%, at least 40%, at least 45%, or at least 50%.
[0079] The dry solids content during saccharification (e.g., total
solids in the cellulosic material) is typically less than about 30
wt. %, 25 wt. %, 20 wt. %, 15 wt. %, 10 wt. %, 7.5 wt. %, 5 wt. %,
2.5 wt. %, 2 wt. %, 1 wt. %, or 0.5 wt. %, such as between 5 and 30
wt. %, such as between 10 and 25 wt. %.
[0080] In an embodiment of the invention the dissolved oxygen
concentration during saccharification is in the range of about
0.5-10% of the saturation level, such as 0.5-5%, such as 0.5-4%,
such as 0.5-3%, such as 0.5-2%, such as 1-5%, such as 1-4%, such as
1-3%, such as 1-2%. In a preferred embodiment the dissolved oxygen
concentration is maintained in the range of about 0.5-10% of the
saturation level, such as 0.5-5%, such as 0.5-4%, such as 0.5-3%,
such as 0.5-2%, such as 1-5%, such as 1-4%, such as 1-3%, such as
1-2% during at least 25%, such as at least 50%, such as at least
75% of the saccharification period.
[0081] Oxygen is added to the vessel in order to achieve the
desired concentration of dissolved oxygen during saccharification.
Maintaining the dissolved oxygen level within a desired range can
be accomplished by aeration of the vessel, tank or the like by
adding compressed air through a diffuser or sparger, or by other
known methods of aeration. The aeration rate can be controlled on
the basis of feedback from a dissolved oxygen sensor placed in the
vessel/tank, or the system can run at a constant rate without
feedback control. In the case of a hydrolysis train consisting of a
plurality of vessels/tanks connected in series, aeration can be
implemented in one or more or all of the vessels/tanks. Oxygen
aeration systems are well known in the art. According to the
invention any suitable aeration system may be used. Commercial
aeration systems are designed by, e.g., Chemineer, Derby, England,
and build by, e.g., Paul Mueller Company, MO, USA.
Methods of Producing Fermentation Products from Cellulosic
Materials
[0082] In another aspect the invention relates to methods of
producing fermentation products from cellulosic material,
comprising:
[0083] (a) subjecting the cellulosic material to a cellulolytic
enzyme composition and a GH61 polypeptide in the presence of
dissolved oxygen at a concentration in the range of 0.5-10% of the
saturation level;
[0084] (b) fermenting the saccharified cellulosic material with one
or more fermenting microorganisms; and
[0085] (c) optionally recovering the fermentation product from
(b).
[0086] In another aspect the invention relates methods of producing
fermentation products from cellulosic material, comprising:
[0087] (a) subjecting the cellulosic material to a cellulolytic
enzyme composition, a GH61 polypeptide, and a catalase in the
presence of dissolved oxygen at a concentration in the range of
0.5-10% of the saturation level;
[0088] (b) fermenting the saccharified cellulosic material with one
or more fermenting microorganisms; and
[0089] (c) optionally recovering the fermentation product from
(b).
[0090] During fermentation, the sugars produced in the
saccharification process are converted into a desired product.
Fermentable sugars may be converted to many useful desired
substances, e.g., fuel, potable ethanol, and/or fermentation
products (e.g., acids, alcohols, ketones, gases, and the like).
Other sugars may be used in processes for producing syrups (e.g.,
High Fructose Corn Syrups (HFCS) and/or plastics (e.g.,
polyethylene, polystyrene, and polypropylene), polylactic acid
(e.g., for producing PET) and more.
[0091] Saccharification and fermentation may be carried out
separately or simultaneously. This includes, but is not limited to,
separate hydrolysis and fermentation (SHF); simultaneous
saccharification and fermentation (SSF); simultaneous
saccharification and cofermentation (SSCF); hybrid hydrolysis and
fermentation (HHF); separate hydrolysis and co-fermentation (SHCF);
hybrid hydrolysis and co-fermentation (HHCF); and direct microbial
conversion (DMC). SHF uses separate steps to first enzymatically
saccharify (hydrolyze) cellulosic material to fermentable sugars,
e.g., glucose, cellobiose, cellotriose, and pentose sugars, and
then ferment the fermentable sugars to ethanol. In SSF, the
enzymatic saccharification of cellulosic materials and the
fermentation of sugars to, e.g., ethanol are combined in one step
(Philippidis, G. P., 1996, Cellulose bioconversion technology, in
Handbook on Bioethanol: Production and Utilization, Wyman, C. E.,
ed., Taylor & Francis, Washington, D.C., 179-212). SSCF
involves the cofermentation of multiple sugars (Sheehan and Himmel,
1999, Enzymes, energy and the environment: A strategic perspective
on the U.S. Department of Energy's research and development
activities for bioethanol, Biotechnol. Prog. 15: 817-827). HHF
involves a separate saccharification (hydrolysis) step, and in
addition a simultaneous saccharification and fermentation step,
which can be carried out in the same reactor. The steps in an HHF
process can be carried out at different temperatures, i.e., high
temperature enzymatic saccharification followed by SSF at a lower
temperature that the fermentation strain can tolerate. DMC combines
all three processes (enzyme production, hydrolysis, and
fermentation) in one or more (several) steps where the same
organism is used to produce the enzymes for conversion of the
cellulosic material to fermentable sugars and to convert the
fermentable sugars into a final product (Lynd et al., 2002,
Microbial cellulose utilization: Fundamentals and biotechnology,
Microbiol. Mol. Biol. Reviews 66: 506-577). It is understood herein
that any method known in the art comprising pretreatment, enzymatic
hydrolysis (saccharification), fermentation, or a combination
thereof, can be used for practicing the methods of the present
invention.
[0092] A conventional apparatus can include a fed-batch stirred
reactor, a batch stirred reactor, a continuous flow stirred reactor
with ultrafiltration, and/or a continuous plug-flow column reactor
(Fernanda de Castilhos Corazza et al., 2003, Optimal control in
fed-batch reactor for the cellobiose hydrolysis, Acta Scientiarum.
Technology 25: 33-38; Gusakov and Sinitsyn, 1985, Kinetics of the
enzymatic hydrolysis of cellulose: 1. A mathematical model for a
batch reactor process, Enz. Microb. Technol. 7: 346-352), an
attrition reactor (Ryu and Lee, 1983, Bioconversion of waste
cellulose by using an attrition bioreactor, Biotechnol. Bioeng. 25:
53-65), or a reactor with intensive stirring induced by an
electromagnetic field (Gusakov et al., 1996, Enhancement of
enzymatic cellulose saccharification using a novel type of
bioreactor with intensive stirring induced by electromagnetic
field, Appl. Biochem. Biotechnol. 56: 141-153). Additional reactor
types include: fluidized bed, upflow blanket, immobilized, and
extruder type reactors for hydrolysis and/or fermentation.
[0093] Cellulosic Material.
[0094] The cellulosic material may be any biomass material. In a
preferred embodiment the cellulosic material has been pretreated,
e.g., by chemical and/or physical pretreatment, such as by dilute
acid and/or steam explosion pretreatment. Examples of suitable
pretreatments can be found in the "Pretreatment"-section below. The
cellulosic material may be pretreated corn stover (PCS), such as
dilute acid pretreated corn stover. The cellulosic material may
also be unwashed, such as unwashed pretreated corn stover
(uwPCS).
[0095] Pretreatment.
[0096] Pretreated cellulosic material may be, e.g., pretreated by a
chemical pretreatment, a physical pretreatment, or a chemical
pretreatment and a physical pretreatment, as described below. In
one aspect, the pretreated cellulosic material has been pretreated
by a chemical pretreatment. In another aspect, the pretreated
cellulosic material has been pretreated by physical pretreatment.
In another aspect, the pretreated cellulosic material has been
pretreated by a chemical pretreatment and a physical pretreatment.
In some aspects, the pretreated cellulosic material is pretreated
corn stover (PCS).
[0097] Any suitable pretreatment process known in the art can be
used to disrupt plant cell wall components of cellulosic material
(Chandra et al., 2007, Substrate pretreatment: The key to effective
enzymatic hydrolysis of lignocellulosics?, Adv. Biochem.
Engin./Biotechnol. 108: 67-93; Galbe and Zacchi, 2007, Pretreatment
of lignocellulosic materials for efficient bioethanol production,
Adv. Biochem. Engin./Biotechnol. 108: 41-65; Hendriks and Zeeman,
2009, Pretreatments to enhance the digestibility of lignocellulosic
biomass, Bioresource Technol. 100: 10-18; Mosier et al., 2005,
Features of promising technologies for pretreatment of
lignocellulosic biomass, Bioresource Technol. 96: 673-686;
Taherzadeh and Karimi, 2008, Pretreatment of lignocellulosic wastes
to improve ethanol and biogas production: A review, Int. J. Mol.
Sci. 9: 1621-1651; Yang and Wyman, 2008, Pretreatment: the key to
unlocking low-cost cellulosic ethanol, Biofuels Bioproducts and
Biorefining-Biofpr. 2: 26-40).
[0098] The cellulosic material can also be subjected to particle
size reduction, pre-soaking, wetting, washing, or conditioning
prior to pretreatment using methods known in the art.
[0099] Conventional pretreatments include, but are not limited to,
steam pretreatment (with or without explosion), dilute acid
pretreatment, hot water pretreatment, alkaline pretreatment, lime
pretreatment, wet oxidation, wet explosion, ammonia fiber
explosion, organosolv pretreatment, and biological pretreatment.
Additional pretreatments include ammonia percolation, ultrasound,
electroporation, microwave, supercritical CO.sub.2, supercritical
H.sub.2O, ozone, and gamma irradiation pretreatments.
[0100] The cellulosic material can be pretreated before hydrolysis
and/or fermentation. Pretreatment is preferably performed prior to
the hydrolysis. Alternatively, the pretreatment can be carried out
simultaneously with enzyme hydrolysis to release fermentable
sugars, such as glucose, xylose, and/or cellobiose. In most cases
the pretreatment step itself results in some conversion of
cellulosic material to fermentable sugars (even in absence of
enzymes).
[0101] Steam Pretreatment.
[0102] In steam pretreatment, the cellulosic material is heated to
disrupt the plant cell wall components, including lignin,
hemicellulose, and cellulose to make the cellulose and other
fractions, e.g., hemicellulose, accessible to enzymes. The
cellulosic material is passed to or through a reaction vessel where
steam is injected to increase the temperature to the required
temperature and pressure and is retained therein for the desired
reaction time. Steam pretreatment may be performed at
140-230.degree. C., e.g., 160-200.degree. C., or 170-190.degree.
C., where the optimal temperature range depends on any addition of
a chemical catalyst. The residence time for the steam pretreatment
may be 1-15 minutes, e.g., 3-12 minutes, or 4-10 minutes, where the
optimal residence time depends on temperature range and any
addition of a chemical catalyst. Steam pretreatment allows for
relatively high solids loadings, so that cellulosic material is
generally only moist during the pretreatment. Steam pretreatment is
often combined with an explosive discharge of the material after
the pretreatment, which is known as steam explosion, that is, rapid
flashing to atmospheric pressure and turbulent flow of the material
to increase the accessible surface area by fragmentation (Duff and
Murray, 1996, Bioresource Technology 855: 1-33; Galbe and Zacchi,
2002, Appl. Microbiol. Biotechnol. 59: 618-628; U.S. Patent
Application No. 2002/0164730). During steam pretreatment,
hemicellulose acetyl groups are cleaved and the resulting acid
autocatalyzes partial hydrolysis of the hemicellulose to
hemicellulose monosaccharides and hemicellulose oligosaccharides,
which become more solubilized. Lignin is removed to only a limited
extent. The resulting liquor primarily contains dissolved
hemicellulosic material (e.g., hemicellulose monosaccharides and
hemicellulose oligosaccharides), whereas the remaining solids
primarily consists of cellulosic material.
[0103] A catalyst such as H.sub.2SO.sub.4 or SO.sub.2 (typically
0.3 to 3% w/w) is often added prior to steam pretreatment, which
decreases the time and temperature, increases the recovery, and
improves enzymatic hydrolysis (Ballesteros et al., 2006, Appl.
Biochem. Biotechnol. 129-132: 496-508; Varga et al., 2004, Appl.
Biochem. Biotechnol. 113-116: 509-523; Sassner et al., 2006, Enzyme
Microb. Technol. 39: 756-762).
[0104] Chemical Pretreatment.
[0105] The term "chemical treatment" refers to any chemical
pretreatment that promotes the separation and/or release of
cellulose, hemicellulose, and/or lignin. Examples of suitable
chemical pretreatment processes include, for example, dilute acid
pretreatment, lime pretreatment, wet oxidation, ammonia
fiber/freeze explosion (AFEX), ammonia percolation (APR), and
organosolv pretreatments.
[0106] In dilute acid pretreatment, cellulosic material is mixed
with dilute acid, typically H.sub.2SO.sub.4, and water to form a
slurry, heated by steam to the desired temperature, and after a
residence time flashed to atmospheric pressure. The dilute acid
pretreatment can be performed with a number of reactor designs,
e.g., plug-flow reactors, counter-current reactors, or continuous
counter-current shrinking bed reactors (Duff and Murray, 1996,
supra; Schell et al., 2004, Bioresource Technol. 91: 179-188; Lee
et al., 1999, Adv. Biochem. Eng. Biotechnol. 65: 93-115).
[0107] Several methods of pretreatment under alkaline conditions
can also be used. These alkaline pretreatments include, but are not
limited to, lime pretreatment, wet oxidation, ammonia percolation
(APR), and ammonia fiber/freeze explosion (AFEX).
[0108] Lime pretreatment is performed with calcium carbonate,
sodium hydroxide, or ammonia at low temperatures of 85-150.degree.
C. and residence times from 1 hour to several days (Wyman et al.,
2005, Bioresource Technol. 96: 1959-1966; Mosier et al., 2005,
Bioresource Technol. 96: 673-686). WO 2006/110891, WO 2006/110899,
WO 2006/110900, and WO 2006/110901 disclose pretreatment methods
using ammonia.
[0109] Wet oxidation is a thermal pretreatment performed typically
at 180-200.degree. C. for 5-15 minutes with addition of an
oxidative agent such as hydrogen peroxide or over-pressure of
oxygen (Schmidt and Thomsen, 1998, Bioresource Technol. 64:
139-151; Palonen et al., 2004, Appl. Biochem. Biotechnol. 117:
1-17; Varga et al., 2004, Biotechnol. Bioeng. 88: 567-574; Martin
et al., 2006, J. Chem. Technol. Biotechnol. 81: 1669-1677). The
pretreatment is performed at preferably 1-40% dry matter, more
preferably 2-30% dry matter, and most preferably 5-20% dry matter,
and often the initial pH is increased by the addition of alkali
such as sodium carbonate.
[0110] A modification of the wet oxidation pretreatment method,
known as wet explosion (combination of wet oxidation and steam
explosion), can handle dry matter up to 30%. In wet explosion, the
oxidizing agent is introduced during pretreatment after a certain
residence time. The pretreatment is then ended by flashing to
atmospheric pressure (WO 2006/032282).
[0111] Ammonia fiber explosion (AFEX) involves treating cellulosic
material with liquid or gaseous ammonia at moderate temperatures
such as 90-100.degree. C. and high pressure such as 17-20 bar for
5-10 minutes, where the dry matter content can be as high as 60%
(Gollapalli et al., 2002, Appl. Biochem. Biotechnol. 98: 23-35;
Chundawat et al., 2007, Biotechnol. Bioeng. 96: 219-231; Alizadeh
et al., 2005, Appl. Biochem. Biotechnol. 121: 1133-1141; Teymouri
et al., 2005, Bioresource Technol. 96: 2014-2018). AFEX
pretreatment results in the depolymerization of cellulose and
partial hydrolysis of hemicellulose. Lignin-carbohydrate complexes
are cleaved.
[0112] Organosolv pretreatment delignifies cellulosic material by
extraction using aqueous ethanol (40-60% ethanol) at
160-200.degree. C. for 30-60 minutes (Pan et al., 2005, Biotechnol.
Bioeng. 90: 473-481; Pan et al., 2006, Biotechnol. Bioeng. 94:
851-861; Kurabi et al., 2005, Appl. Biochem. Biotechnol. 121:
219-230). Sulphuric acid is usually added as a catalyst. In
organosolv pretreatment, the majority of hemicellulose is
removed.
[0113] Other examples of suitable pretreatment methods are
described by Schell et al., 2003, Appl. Biochem. and Biotechnol.
105-108: 69-85, and Mosier et al., 2005, Bioresource Technology 96:
673-686, and U.S. Published Application 2002/0164730.
[0114] In one aspect, the chemical pretreatment is carried out as
an acid treatment, such as a continuous dilute and/or mild acid
treatment. The acid is may be sulfuric acid, but other acids can
also be used, such as acetic acid, citric acid, nitric acid,
phosphoric acid, tartaric acid, succinic acid, hydrogen chloride,
or mixtures thereof. Mild acid treatment is conducted in the pH
range of preferably 1-5, more preferably 1-4, and most preferably
1-3. In one aspect, the acid concentration is in the range of 0.01
to 20 wt. % acid, preferably 0.05 to 10 wt. % acid, more preferably
0.1 to 5 wt. % acid, and most preferably 0.2 to 2.0 wt. % acid. The
acid is contacted with biomass material and held at a temperature
in the range of preferably 160-220.degree. C., and more preferably
165-195.degree. C., for periods ranging from seconds to minutes,
e.g., 1 second to 60 minutes.
[0115] In another aspect, pretreatment is carried out as an ammonia
fiber explosion step (AFEX pretreatment step).
[0116] In another aspect, pretreatment takes place in an aqueous
slurry. In one aspect, cellulosic material is present during
pretreatment in amounts preferably between 10-80 wt. %, e.g.,
between 20-70 wt. %, or between 30-60 wt. %, such as around 50 wt.
%. The pretreated cellulosic material can be unwashed or washed
using any method known in the art, e.g., washed with water.
[0117] Mechanical Pretreatment or Physical Pretreatment.
[0118] The term "mechanical pretreatment" or "physical
pretreatment" refers to any pretreatment that promotes size
reduction of particles. For example, such pretreatment can involve
various types of grinding or milling (e.g., dry milling, wet
milling, or vibratory ball milling).
[0119] The cellulosic material can be pretreated both physically
(mechanically) and chemically. Mechanical or physical pretreatment
can be coupled with steaming/steam explosion, hydrothermolysis,
dilute or mild acid treatment, high temperature, high pressure
treatment, irradiation (e.g., microwave irradiation), or
combinations thereof. In one aspect, high pressure means pressure
in the range of preferably about 100 to about 400 psi, more
preferably about 150 to about 250 psi. In another aspect, high
temperature means temperatures in the range of about 100 to about
300.degree. C., preferably about 140 to about 200.degree. C. In a
preferred aspect, mechanical or physical pretreatment is performed
in a batch-process using a steam gun hydrolyzer system that uses
high pressure and high temperature as defined above, e.g., a Sunds
Hydrolyzer available from Sunds Defibrator AB, Sweden. The physical
and chemical pretreatments can be carried out sequentially or
simultaneously, as desired.
[0120] Accordingly, in a preferred aspect, the cellulosic material
is subjected to physical (mechanical) or chemical pretreatment, or
any combination thereof, to promote the separation and/or release
of cellulose, hemicellulose, and/or lignin.
[0121] Biological Pretreatment:
[0122] The term "biological pretreatment" refers to any biological
pretreatment that promotes the separation and/or release of
cellulose, hemicellulose, and/or lignin from biomass material.
Biological pretreatment techniques can involve applying
lignin-solubilizing microorganisms (see, for example, Hsu, T.-A.,
1996, Pretreatment of biomass, in Handbook on Bioethanol:
Production and Utilization, Wyman, C. E., ed., Taylor &
Francis, Washington, D.C., 179-212; Ghosh and Singh, 1993,
Physicochemical and biological treatments for enzymatic/microbial
conversion of cellulosic biomass, Adv. Appl. Microbiol. 39:
295-333; McMillan, J. D., 1994, Pretreating lignocellulosic
biomass: a review, in Enzymatic Conversion of Biomass for Fuels
Production, Himmel, M. E., Baker, J. O., and Overend, R. P., eds.,
ACS Symposium Series 566, American Chemical Society, Washington,
D.C., chapter 15; Gong, C. S., Cao, N. J., Du, J., and Tsao, G. T.,
1999, Ethanol production from renewable resources, in Advances in
Biochemical Engineering/Biotechnology, Scheper, T., ed.,
Springer-Verlag Berlin Heidelberg, Germany, 65: 207-241; Olsson and
Hahn-Hagerdal, 1996, Fermentation of lignocellulosic hydrolysates
for ethanol production, Enz. Microb. Tech. 18: 312-331; and
Vallander and Eriksson, 1990, Production of ethanol from
lignocellulosic materials: State of the art, Adv. Biochem.
Eng./Biotechnol. 42: 63-95).
[0123] Fermentation.
[0124] The fermentable sugars obtained from the saccharifying
cellulosic material in accordance with the invention can be
fermented by one or more (several) fermenting microorganisms
capable of fermenting the sugars (e.g., glucose, xylose) directly
or indirectly into a desired fermentation product (e.g.,
ethanol).
[0125] "Fermentation" or "fermentation process" refers to any
fermentation process or any process comprising a fermentation step.
Fermentation processes also include fermentation processes used in
the consumable alcohol industry (e.g., beer and wine), dairy
industry (e.g., fermented dairy products), leather industry, and
tobacco industry. The fermentation conditions depend on the desired
fermentation product and fermenting organism and can easily be
determined by one skilled in the art.
[0126] In the fermentation step, sugars, released from cellulosic
material as a result of the enzymatic saccharification, are
fermented to a product, e.g., ethanol, by a fermenting organism,
such as yeast. Saccharification and fermentation can be separate or
simultaneous, as described herein.
[0127] Any suitable cellulosic material saccharified according to
the invention can be used in the fermentation step in practicing
the present invention. The material is generally selected based on
the desired fermentation product, i.e., the substance to be
obtained from the fermentation, and the process employed.
[0128] The term "fermentation medium" is understood herein to refer
to a medium before the fermenting microorganism(s) is(are) added,
such as, a medium resulting from saccharification, as well as a
medium used in, e.g., a simultaneous saccharification and
fermentation process (SSF).
[0129] "Fermenting microorganism" refers to any microorganism,
including bacterial and fungal organisms, suitable for use in a
desired fermentation process to produce a fermentation product. The
fermenting organism can be hexose and/or pentose fermenting
organisms, or a combination thereof. Both hexose and pentose
fermenting organisms are well known in the art. Suitable fermenting
microorganisms are able to ferment, i.e., convert, sugars, such as
glucose, xylose, xylulose, arabinose, maltose, mannose, galactose,
and/or oligosaccharides, directly or indirectly into the desired
fermentation product.
[0130] Examples of bacterial and fungal fermenting organisms
producing ethanol are described by Lin et al., 2006, Appl.
Microbiol. Biotechnol. 69: 627-642.
[0131] Examples of fermenting microorganisms that can ferment
C.sub.6 sugars include bacterial and fungal organisms, such as
yeast. Preferred yeast includes strains of Saccharomyces spp.,
preferably Saccharomyces cerevisiae.
[0132] Examples of fermenting organisms that can ferment C.sub.5
sugars include bacterial and fungal organisms, such as yeast.
Preferred C.sub.5 fermenting yeast include strains of Pichia,
preferably Pichia stipitis, such as Pichia stipitis CBS 5773;
strains of Candida, preferably Candida boidinii, Candida brassicae,
Candida sheatae, Candida diddensii, Candida pseudotropicalis, or
Candida utilis.
[0133] Other fermenting organisms include strains of Zymomonas,
such as Zymomonas mobilis; Hansenula, such as Hansenula anomala;
Kluyveromyces, such as K. marxianus, K lactis, K. thermotolerans,
and K. fragilis; Schizosaccharomyces, such as S. pombe; E. coli,
especially E. coli strains that have been genetically modified to
improve the yield of ethanol; Clostridium, such as Clostridium
acetobutylicum, Chlostridium thermocellum, and Chlostridium
phytofermentans; Geobacillus sp.; Thermoanaerobacter, such as
Thermoanaerobacter saccharolyticum; and Bacillus, such as Bacillus
coagulans; Candida, such as C. sonorensis, C. methanosorbosa, C.
diddensiae, C. parapsilosis, C. naedodendra, C. blankii C.
entomophilia, C. brassicae, C. pseudotropicalis, C. boidinii, C.
utilis, and C. scehatae; Klebsiella, such as K. oxytoca.
[0134] In one aspect, the yeast is a Saccharomyces spp. In another
aspect, the yeast is Saccharomyces cerevisiae. In another aspect,
the yeast is Saccharomyces distaticus. In another aspect, the yeast
is Saccharomyces uvarum. In another aspect, the yeast is a
Kluyveromyces. In another aspect, the yeast is Kluyveromyces
marxianus. In another aspect, the yeast is Kluyveromyces fragilis.
In another aspect, the yeast is a Candida. In another aspect, the
yeast is Candida boidinii. In another aspect, the yeast is Candida
brassicae. In another aspect, the yeast is Candida diddensii. In
another aspect, the yeast is Candida pseudotropicalis. In another
aspect, the yeast is Candida utilis. In another aspect, the yeast
is a Clavispora. In another aspect, the yeast is Clavispora
lusitaniae. In another aspect, the yeast is Clavispora opuntiae. In
another aspect, the yeast is a Pachysolen. In another aspect, the
yeast is Pachysolen tannophilus. In another aspect, the yeast is a
Pichia. In another aspect, the yeast is a Pichia stipitis. In
another aspect, the yeast is a Bretannomyces. In another aspect,
the yeast is Bretannomyces clausenii (Philippidis, 1996,
supra).
[0135] Bacteria that can efficiently ferment hexose and pentose to
ethanol include, for example, Zymomonas mobilis, Clostridium
acetobutylicum, Clostridium thermocellum, Clostridium
phytofermentans, Geobacillus sp., Thermoanaerobacter
saccharolyticum, and Bacillus coagulans (Philippidis, 1996,
supra).
[0136] In one aspect, the bacterium is a Zymomonas. In one aspect,
the bacterium is Zymomonas mobilis. In another aspect, the
bacterium is a Clostridium. In another aspect, the bacterium is
Clostridium acetobutylicum. In another aspect, the bacterium is
Clostridium phytofermentan. In another aspect, the bacterium is
Clostridium thermocellum. In another aspect, the bacterium is
Geobacillus sp. In another aspect, the bacterium is
Thermoanaerobacter saccharolyticum. In another aspect, the
bacterium is Bacillus coagulans.
[0137] Commercially available yeast suitable for ethanol production
includes, e.g., ETHANOL RED.TM. yeast (available from
Fermentis/Lesaffre, USA), FALI.TM. (available from Fleischmann's
Yeast, USA), SUPERSTART.TM. and THERMOSACC.TM. fresh yeast
(available from Ethanol Technology, WI, USA), BIOFERM.TM. AFT and
XR (available from NABC--North American Bioproducts Corporation,
GA, USA), GERT STRAND.TM. (available from Gert Strand AB, Sweden),
and FERMIOL.TM. (available from DSM Specialties).
[0138] In one aspect, the fermenting microorganism has been
genetically modified to provide the ability to ferment pentose
sugars, such as xylose utilizing, arabinose utilizing, and xylose
and arabinose co-utilizing microorganisms.
[0139] The cloning of heterologous genes into various fermenting
microorganisms has led to the construction of organisms capable of
converting hexoses and pentoses to ethanol (cofermentation) (Chen
and Ho, 1993, Cloning and improving the expression of Pichia
stipitis xylose reductase gene in Saccharomyces cerevisiae, Appl.
Biochem. Biotechnol. 39-40: 135-147; Ho et al., 1998, Genetically
engineered Saccharomyces yeast capable of effectively cofermenting
glucose and xylose, Appl. Environ. Microbiol. 64: 1852-1859; Kotter
and Ciriacy, 1993, Xylose fermentation by Saccharomyces cerevisiae,
Appl. Microbiol. Biotechnol. 38: 776-783; Walfridsson et al., 1995,
Xylose-metabolizing Saccharomyces cerevisiae strains overexpressing
the TKL1 and TALI genes encoding the pentose phosphate pathway
enzymes transketolase and transaldolase, Appl. Environ. Microbiol.
61: 4184-4190; Kuyper et al., 2004, Minimal metabolic engineering
of Saccharomyces cerevisiae for efficient anaerobic xylose
fermentation: a proof of principle, FEMS Yeast Research 4: 655-664;
Beall et al., 1991, Parametric studies of ethanol production from
xylose and other sugars by recombinant Escherichia coli, Biotech.
Bioeng. 38: 296-303; Ingram et al., 1998, Metabolic engineering of
bacteria for ethanol production, Biotechnol. Bioeng. 58: 204-214;
Zhang et al., 1995, Metabolic engineering of a pentose metabolism
pathway in ethanologenic Zymomonas mobilis, Science 267: 240-243;
Deanda et al., 1996, Development of an arabinose-fermenting
Zymomonas mobilis strain by metabolic pathway engineering, Appl.
Environ. Microbiol. 62: 4465-4470; WO 2003/062430, xylose
isomerase).
[0140] In one aspect, the genetically modified fermenting
microorganism is Saccharomyces cerevisiae. In another aspect, the
genetically modified fermenting microorganism is Zymomonas mobilis.
In another aspect, the genetically modified fermenting
microorganism is Escherichia coli. In another aspect, the
genetically modified fermenting microorganism is Klebsiella
oxytoca. In another aspect, the genetically modified fermenting
microorganism is Kluyveromyces sp.
[0141] It is well known in the art that the organisms described
above can also be used to produce other substances, as described
herein.
[0142] The fermenting microorganism is typically added to
saccharified pretreated cellulosic material and the fermentation
may be performed for about 8 to about 96 hours, such as about 24 to
about 60 hours. The temperature is typically between about
26.degree. C. to about 60.degree. C., in particular about
32.degree. C. or 50.degree. C., and at about pH 3 to about pH 8,
such as around pH 4-5, 6, or 7.
[0143] In one aspect, the yeast and/or another microorganism, is
applied to the saccharified pretreated cellulosic material and then
fermentation is performed for about 12 hours to about 96 hours,
such as 24-60 hours. In one aspect, the temperature is between
about 20.degree. C. to about 60.degree. C., e.g., about 25.degree.
C. to about 50.degree. C., or about 32.degree. C. to about
50.degree. C., and the pH is generally from about pH 3 to about pH
7, e.g., around pH 4-7, such as about pH 5.
[0144] However, some fermenting organisms, e.g., bacteria, have
higher fermentation temperature optima. Yeast or another
microorganism is preferably applied in amounts of approximately
10.sup.5 to 10.sup.12, e.g., from approximately 10.sup.7 to
10.sup.10, especially approximately 2.times.10.sup.8 viable cell
count per mL of fermentation broth. Further guidance in respect of
using yeast for fermentation can be found in, e.g., "The Alcohol
Textbook" (Editors K. Jacques, T. P. Lyons and D. R. Kelsall,
Nottingham University Press, United Kingdom 1999), which is hereby
incorporated by reference.
[0145] For ethanol production, following the fermentation, the
fermented slurry may be distilled to extract the ethanol. The
ethanol obtained according to a method of the invention can be used
as, e.g., fuel ethanol, drinking ethanol, i.e., potable neutral
spirits, or industrial ethanol.
[0146] A fermentation stimulator can be used in combination with
any of the methods described herein to further improve the
fermentation process, and in particular, the performance of the
fermenting microorganism, such as, rate enhancement and ethanol
yield. A "fermentation stimulator" refers to stimulators for growth
of the fermenting microorganisms, in particular, yeast. Preferred
fermentation stimulators for growth include vitamins and minerals.
Examples of vitamins include multivitamins, biotin, pantothenate,
nicotinic acid, meso-inositol, thiamine, pyridoxine,
para-aminobenzoic acid, folic acid, riboflavin, and Vitamins A, B,
C, D, and E. See, for example, Alfenore et al., Improving ethanol
production and viability of Saccharomyces cerevisiae by a vitamin
feeding strategy during fed-batch process, Springer-Verlag (2002),
which is hereby incorporated by reference. Examples of minerals
include minerals and mineral salts that can supply nutrients
comprising P, K, Mg, S, Ca, Fe, Zn, Mn, and Cu.
[0147] Fermentation Products:
[0148] The fermentation product can be any substance derived from
fermentation. The fermentation product can, without limitation, be
an alcohol (e.g., arabinitol, butanol, ethanol, glycerol, methanol,
1,3-propanediol, sorbitol, and xylitol); an organic acid (e.g.,
acetic acid, acetonic acid, adipic acid, ascorbic acid, citric
acid, 2,5-diketo-D-gluconic acid, formic acid, fumaric acid,
glucaric acid, gluconic acid, glucuronic acid, glutaric acid,
3-hydroxypropionic acid, itaconic acid, lactic acid, malic acid,
malonic acid, oxalic acid, oxaloacetic acid, propionic acid,
succinic acid, and xylonic acid); a ketone (e.g., acetone); an
amino acid (e.g., aspartic acid, glutamic acid, glycine, lysine,
serine, and threonine); an alkane (e.g., pentane, hexane, heptane,
octane, nonane, decane, undecane, and dodecane), a cycloalkane
(e.g., cyclopentane, cyclohexane, cycloheptane, and cyclooctane),
an alkene (e.g., pentene, hexene, heptene, and octene); and a gas
(e.g., methane, hydrogen (H.sub.2), carbon dioxide (CO.sub.2), and
carbon monoxide (CO)). The fermentation product can also be protein
as a high value product.
[0149] In one aspect, the fermentation product is an alcohol. It
will be understood that the term "alcohol" encompasses a substance
that contains one or more hydroxyl moieties. In one aspect, the
alcohol is arabinitol. In another aspect, the alcohol is butanol.
In another aspect, the alcohol is ethanol. In another aspect, the
alcohol is glycerol. In another aspect, the alcohol is methanol. In
another aspect, the alcohol is 1,3-propanediol. In another aspect,
the alcohol is sorbitol. In another aspect, the alcohol is xylitol.
See, for example, Gong et al., 1999, Ethanol production from
renewable resources, in Advances in Biochemical
Engineering/Biotechnology, Scheper, T., ed., Springer-Verlag Berlin
Heidelberg, Germany, 65: 207-241; Silveira and Jonas, 2002, The
biotechnological production of sorbitol, Appl. Microbiol.
Biotechnol. 59: 400-408; Nigam and Singh, 1995, Processes for
fermentative production of xylitol--a sugar substitute, Process
Biochemistry 30(2): 117-124; Ezeji et al., 2003, Production of
acetone, butanol and ethanol by Clostridium beijerinckii BA101 and
in situ recovery by gas stripping, World Journal of Microbiology
and Biotechnology 19(6): 595-603.
[0150] In another aspect, the fermentation product is an organic
acid. In one aspect, the organic acid is acetic acid. In another
aspect, the organic acid is acetonic acid. In another aspect, the
organic acid is adipic acid. In another aspect, the organic acid is
ascorbic acid. In another aspect, the organic acid is citric acid.
In another aspect, the organic acid is 2,5-diketo-D-gluconic acid.
In another aspect, the organic acid is formic acid. In another
aspect, the organic acid is fumaric acid. In another aspect, the
organic acid is glucaric acid. In another aspect, the organic acid
is gluconic acid. In another aspect, the organic acid is glucuronic
acid. In another aspect, the organic acid is glutaric acid. In
another aspect, the organic acid is 3-hydroxypropionic acid. In
another aspect, the organic acid is itaconic acid. In another
aspect, the organic acid is lactic acid. In another aspect, the
organic acid is malic acid. In another aspect, the organic acid is
malonic acid. In another aspect, the organic acid is oxalic acid.
In another aspect, the organic acid is propionic acid. In another
aspect, the organic acid is succinic acid. In another aspect, the
organic acid is xylonic acid. See, for example, Chen and Lee, 1997,
Membrane-mediated extractive fermentation for lactic acid
production from cellulosic biomass, Appl. Biochem. Biotechnol.
63-65: 435-448.
[0151] In another aspect, the fermentation product is a ketone. It
will be understood that the term "ketone" encompasses a substance
that contains one or more ketone moieties. In another aspect, the
ketone is acetone. See, for example, Qureshi and Blaschek, 2003,
supra.
[0152] In another aspect, the fermentation product is an amino
acid. In one aspect, the amino acid is aspartic acid. In another
aspect, the amino acid is glutamic acid. In another aspect, the
amino acid is glycine. In another aspect, the amino acid is lysine.
In another aspect, the amino acid is serine. In another aspect, the
amino acid is threonine. See, for example, Richard and Margaritis,
2004, Empirical modeling of batch fermentation kinetics for
poly(glutamic acid) production and other microbial biopolymers,
Biotechnology and Bioengineering 87(4): 501-515.
[0153] In another aspect, the fermentation product is an alkane.
The alkane can be an unbranched or a branched alkane. In one
aspect, the alkane is pentane. In another aspect, the alkane is
hexane. In another aspect, the alkane is heptane. In another
aspect, the alkane is octane. In another aspect, the alkane is
nonane. In another aspect, the alkane is decane. In another aspect,
the alkane is undecane. In another aspect, the alkane is
dodecane.
[0154] In another aspect, the fermentation product is a
cycloalkane. In one aspect, the cycloalkane is cyclopentane. In
another aspect, the cycoalkane is cyclohexane. In another aspect,
the cycloalkane is cycloheptane. In another aspect, the cycloalkane
is cyclooctane.
[0155] In another aspect, the fermentation product is an alkene.
The alkene can be an unbranched or a branched alkene. In one
aspect, the alkene is pentene. In another aspect, the alkene is
hexene. In another aspect, the alkene is heptene. In another
aspect, the alkene is octene.
[0156] In one aspect, the fermentation product is isoprene. In
another aspect, the fermentation product is polyketide.
[0157] In another aspect, the fermentation product is a gas. In one
aspect, the gas is methane. In another aspect, the gas is H.sub.2.
In another aspect, the gas is CO.sub.2. In another aspect, the gas
is CO. See, for example, Kataoka et al., 1997, Studies on hydrogen
production by continuous culture system of hydrogen-producing
anaerobic bacteria, Water Science and Technology 36(6-7): 41-47;
and Gunaseelan, 1997, Anaerobic digestion of biomass for methane
production: A review, Biomass and Bioenergy 13(1-2): 83-114.
[0158] Recovery.
[0159] The fermentation product(s) may optionally be recovered from
the fermentation medium using any method known in the art
including, but not limited to, chromatography, electrophoretic
procedures, differential solubility, distillation, or extraction.
For example, alcohol is separated from the fermented sugar cane
trash and purified by conventional methods of distillation. Ethanol
with a purity of up to about 96 vol. % can be obtained, which can
be used as, for example, fuel ethanol, drinking ethanol, i.e.,
potable neutral spirits, or industrial ethanol.
Enzymes
[0160] The enzyme(s) and polypeptides described below are to be
used in an "effective amount" in processes of the present
invention. Below should be read in context of the enzyme disclosure
in the "Definitions"-section above.
Cellulolytic Enzyme Compositions Used for Saccharification
[0161] The cellulolytic enzyme compositions can comprise any
protein useful in degrading the cellulosic material. The
cellulolytic enzyme composition used for saccharification may be of
any origin, such as microbial origin, such as eukaryotic origin,
such as fungal origin, e.g., filamentous fungal origin.
[0162] In one aspect, the cellulolytic enzyme composition comprises
or further comprises one or more (e.g., several) proteins selected
from the group consisting of a cellulase, a hemicellulase, an
esterase, an expansin, a ligninolytic enzyme, an oxidoreductase, a
pectinase, a protease, and a swollenin. In another aspect, the
cellulase is preferably one or more (e.g., several) enzymes
selected from the group consisting of an endoglucanase, a
cellobiohydrolase, and a beta-glucosidase. In another aspect, the
hemicellulase is preferably one or more (e.g., several) enzymes
selected from the group consisting of an acetylmannan esterase, an
acetylxylan esterase, an arabinanase, an arabinofuranosidase, a
coumaric acid esterase, a feruloyl esterase, a galactosidase, a
glucuronidase, a glucuronoyl esterase, a mannanase, a mannosidase,
a xylanase, and a xylosidase. In another aspect, the oxidoreductase
is a catalase, a laccase, or a peroxidase.
[0163] In another aspect, the cellulolytic enzyme composition
comprises one or more (e.g., several) cellulolytic enzymes. In
another aspect, the cellulolytic enzyme composition comprises or
further comprises one or more (e.g., several) hemicellulolytic
enzymes. In another aspect, the cellulolytic enzyme composition
comprises one or more (e.g., several) cellulolytic enzymes and one
or more (e.g., several) hemicellulolytic enzymes. In another
aspect, the cellulolytic enzyme composition comprises an
endoglucanase. In another aspect, the cellulolytic enzyme
composition comprises a cellobiohydrolase. In another aspect, the
cellulolytic enzyme composition comprises a beta-glucosidase. In
another aspect, the cellulolytic enzyme composition comprises an
endoglucanase and a cellobiohydrolase. In another aspect, the
cellulolytic enzyme composition comprises an endoglucanase and a
cellobiohydrolase I, a cellobiohydrolase II, or a combination of a
cellobiohydrolase I and a cellobiohydrolase II. In another aspect,
the cellulolytic enzyme composition comprises an endoglucanase and
a beta-glucosidase. In another aspect, the cellulolytic enzyme
composition comprises a beta-glucosidase and a cellobiohydrolase.
In another aspect, the cellulolytic enzyme composition comprises a
beta-glucosidase and a cellobiohydrolase I, a cellobiohydrolase II,
or a combination of a cellobiohydrolase I and a cellobiohydrolase
II. In another aspect, the cellulolytic enzyme composition
comprises an endoglucanase, a beta-glucosidase, and a
cellobiohydrolase. In another aspect, the cellulolytic enzyme
composition comprises an endoglucanase, a beta-glucosidase, and a
cellobiohydrolase I, a cellobiohydrolase II, or a combination of a
cellobiohydrolase I and a cellobiohydrolase II.
[0164] In another aspect, the cellulolytic enzyme composition
comprises an acetylmannan esterase. In another aspect, the
cellulolytic enzyme composition comprises an acetylxylan esterase.
In another aspect, the cellulolytic enzyme composition comprises an
arabinanase (e.g., alpha-L-arabinanase). In another aspect, the
cellulolytic enzyme composition comprises an arabinofuranosidase
(e.g., alpha-L-arabinofuranosidase). In another aspect, the
cellulolytic enzyme composition comprises a coumaric acid esterase.
In another aspect, the enzyme composition comprises a feruloyl
esterase. In another aspect, the cellulolytic enzyme composition
comprises a galactosidase (e.g., alpha-galactosidase and/or
beta-galactosidase). In another aspect, the cellulolytic enzyme
composition comprises a glucuronidase (e.g.,
alpha-D-glucuronidase). In another aspect, the cellulolytic enzyme
composition comprises a glucuronoyl esterase. In another aspect,
the cellulolytic enzyme composition comprises a mannanase. In
another aspect, the cellulolytic enzyme composition comprises a
mannosidase (e.g., beta-mannosidase). In another aspect, the
cellulolytic enzyme composition comprises a xylanase. In an
embodiment, the xylanase is a Family 10 xylanase. In another
embodiment, the xylanase is a Family 11 xylanase. In another
aspect, the cellulolytic enzyme composition comprises a xylosidase
(e.g., beta-xylosidase).
[0165] In another aspect, the cellulolytic enzyme composition
comprises a CIP. In another aspect, the cellulolytic enzyme
composition comprises an esterase. In another aspect, the
cellulolytic enzyme composition comprises an expansin. In another
aspect, the cellulolytic enzyme composition comprises a
ligninolytic enzyme. In an embodiment, the ligninolytic enzyme is a
manganese peroxidase. In another embodiment, the ligninolytic
enzyme is a lignin peroxidase. In another embodiment, the
ligninolytic enzyme is a H.sub.2O.sub.2-producing enzyme. In
another aspect, the cellulolytic enzyme composition comprises a
pectinase. In another aspect, the cellulolytic enzyme composition
comprises an oxidoreductase. In another embodiment, the
oxidoreductase is a laccase. In another embodiment, the
oxidoreductase is a peroxidase. In another aspect, the enzyme
composition comprises a protease. In another aspect, the enzyme
composition comprises a swollenin.
[0166] In an embodiment the cellulolytic enzyme composition is
derived or isolated from a strain of Trichoderma, such as a strain
of Trichoderma reesei; a strain of Humicola, such as a strain of
Humicola insolens, and/or a strain of Chrysosporium, such as a
strain of Chrysosporium lucknowense. In a preferred embodiment the
cellulolytic enzyme composition is derived or isolated from a
strain of Trichoderma reesei.
[0167] Examples of Trichoderma reseei cellulolytic enzyme
compositions with recombinantly produced GH61 polypeptide are
described in WO 2008/151079 (Novozymes) and WO 2013/028928
(Novozymes) which are both hereby incorporated by reference.
Examples of suitable GH61 polypeptides can be found in the "GH61
polypeptide"-section below.
[0168] The cellulolytic enzyme composition may further comprise one
or more enzymes selected from the group consisting of: esterases,
expansins, hemicellulases, laccases, ligninolytic enzymes,
pectinases, peroxidases, proteases, and swollenins.
[0169] The optimum amount of the cellulolytic enzyme composition
depends on several factors including, but not limited to, the
mixture of component enzymes, the cellulosic material, the
concentration of the cellulosic material, the pretreatment(s) of
the cellulosic material, temperature, time, pH, and inclusion of
fermenting organism (e.g., yeast).
[0170] The cellulolytic enzyme composition may be added in an
amount of about 0.01 to about 50.0 mg, e.g., about 1 to about 25
mg, such as about 2-10 mg, such as about 4 to about 8 mg protein
per g/DS of the cellulosic material.
Beta-Glucosidases
[0171] The cellulolytic enzyme composition used according to the
invention may in one embodiment comprise one or more
beta-glucosidase. The beta-glucosidase may be of any origin, such
as microbial origin, such as eukaryotic origin, such as fungal
origin, e.g., filamentous origin.
[0172] In one embodiment the beta-glucosidase is from a strain of
Aspergillus, such as Aspergillus oryzae, such as the one disclosed
in WO 02/095014 or the fusion protein having beta-glucosidase
activity disclosed in WO 2008/057637 (see e.g., Examples 10-15), or
Aspergillus fumigatus, such as the one disclosed as SEQ ID NO: 2 in
WO 2005/047499 or SEQ ID NO: 5 herein or an Aspergillus fumigatus
beta-glucosidase variant, such as one disclosed in WO 2012/044915,
such as one with the following substitutions: F100D, S283G, N456E,
F512Y (using SEQ ID NO: 5 herein for numbering).
[0173] In another embodiment the beta-glucosidase is derived from a
strain of Penicillium, such as a strain of the Penicillium
brasilianum disclosed as SEQ ID NO: 2 in WO 2007/019442, or a
strain of Trichoderma, such as a strain of Trichoderma reesei.
[0174] In an embodiment beta-glucosidase is an Aspergillus
fumigatus beta-glucosidase or homolog thereof selected from the
group consisting of:
[0175] (i) a beta-glucosidase comprising the mature polypeptide of
SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 5 herein;
[0176] (ii) a beta-glucosidase comprising an amino acid sequence
having at least 70%, e.g., at least 75%, at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99% identity to the mature polypeptide of SEQ ID NO: 2
in WO 2005/047499 or SEQ ID NO: 5 herein;
[0177] (iii) a beta-glucosidase encoded by a polynucleotide
comprising a nucleotide sequence having at least 70%, e.g., at
least 75%, at least 80%, at least 85%, at least 90%, at leasy 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% identity to the
mature polypeptide coding sequence of SEQ ID NO: 1 in WO
2005/047499; and
[0178] (iv) a beta-glucosidase encoded by a polynucleotide that
hybridizes under medium, high stringency conditions, or very high
stringency conditions, with the mature polypeptide coding sequence
of SEQ ID NO: 1 in WO 2005/047499 or the full-length complement
thereof.
[0179] In an embodiment the beta-glucosidase is a variant
comprising a substitution at one or more (several) positions
corresponding to positions 100, 283, 456, and 512 of the mature
polypeptide of SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 5
herein, wherein the variant has beta-glucosidase activity.
[0180] In an embodiment the parent beta-glucosidase of the variant
is (a) a polypeptide comprising the mature polypeptide of SEQ ID
NO: 2 in WO 2005/047499 or SEQ ID NO: 5 herein; (b) a polypeptide
having at least 80% sequence identity to the mature polypeptide of
SEQ ID NO: 5 herein; (c) a polypeptide encoded by a polynucleotide
that hybridizes under low, medium, high or very high stringency
conditions with (i) the mature polypeptide coding sequence of SEQ
ID NO: 1 in WO 2005/047499 (hereby incorporated by reference), (ii)
the cDNA sequence contained in the mature polypeptide coding
sequence of SEQ ID NO: 5, or (iii) the full-length complementary
strand of (i) or (ii); (d) a polypeptide encoded by a
polynucleotide having at least 80% identity to the mature
polypeptide coding sequence of SEQ ID NO: 1 in WO 2005/047499 or
the cDNA sequence thereof; or (e) a fragment of the mature
polypeptide of SEQ ID NO: 2 in WO 2005/047499, which has
beta-glucosidase activity.
[0181] In an embodiment the variant has at least 80%, e.g., at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
but less than 100%, sequence identity to the amino acid sequence of
the parent beta-glucosidase.
[0182] In an embodiment the variant has at least 80%, e.g., at
least 81%, at least 82%, at least 83%, at least 84%, at least 85%,
at least 86%, at least 87%, at least 88%, at least 89%, at least
90%, at least 91%, at least 92%, at least 93%, at least 94%, at
least 95%, at least 96%, at least 97%, at least 98%, at least 99%,
but less than 100% sequence identity to the mature polypeptide of
SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 5 herein.
[0183] In an embodiment the number of substitutions is between 1
and 4, such as 1, 2, 3, or 4 substitutions.
[0184] In an embodiment the variant comprises a substitution at a
position corresponding to position 100, a substitution at a
position corresponding to position 283, a substitution at a
position corresponding to position 456, and/or a substitution at a
position corresponding to position 512.
[0185] In an embodiment the beta-glucosidase variant comprises the
following substitutions: Phe100Asp, Ser283Gly, Asn456Glu, Phe512Tyr
in SEQ ID NO: 2 in WO 2005/047499 or SEQ ID NO: 5 herein.
Endoglucanases
[0186] The cellulolytic enzyme composition used according to the
invention comprises one or more endoglucoanase. The endoglucanase
may be of any origin, such as microbial origin, such as eukaryotic
origin, such as fungal origin, e.g., filamentous origin.
[0187] In an embodiment the endoglucanase(s) may be from a strain
of Trichoderma, such as a strain of Trichoderma reesei; a strain of
Humicola, such as a strain of Humicola insolens, and/or a strain of
Chrysosporium, such as a strain of Chrysosporium lucknowense. In a
preferred embodiment the endoglucoamase is derived from a strain of
Trichoderma reesei.
[0188] Examples of fungal endoglucanases that can be used according
to the present invention include, but are not limited to, a
Trichoderma reesei endoglucanase I (Penttila et al., 1986, Gene 45:
253-263; Trichoderma reesei Cel7B endoglucanase I; GENBANK.TM.
accession no. M15665; Trichoderma reesei endoglucanase II
(Saloheimo, et al., 1988, Gene 63:11-22; Trichoderma reesei Cel5A
endoglucanase II; GENBANK.TM. accession no. M19373; Trichoderma
reesei endoglucanase III (Okada et al., 1988, Appl. Environ.
Microbiol. 64: 555-563; GENBANK.TM. accession no. AB003694;
Trichoderma reesei endoglucanase V (Saloheimo et al., 1994,
Molecular Microbiology 13: 219-228; GENBANK.TM. accession no.
Z33381; Aspergillus aculeatus endoglucanase (Ooi et al., 1990,
Nucleic Acids Research 18: 5884); Aspergillus kawachii
endoglucanase (Sakamoto et al., 1995, Current Genetics 27:
435-439); Erwinia carotovara endoglucanase (Saarilahti et al.,
1990, Gene 90: 9-14); Fusarium oxysporum endoglucanase (GENBANK.TM.
accession no. L29381); Humicola grisea var. thermoidea
endoglucanase (GENBANK.TM. accession no. AB003107); Melanocarpus
albomyces endoglucanase (GENBANK.TM. accession no. MAL515703);
Neurospora crassa endoglucanase (GENBANK.TM. accession no.
XM.sub.--324477); Humicola insolens endoglucanase V; Myceliophthora
thermophila CBS 117.65 endoglucanase basidiomycete CBS 495.95
endoglucanase; basidiomycete CBS 494.95 endoglucanase; Thielavia
terrestris NRRL 8126 CEL6B endoglucanase; Thielavia terrestris NRRL
8126 CEL6C endoglucanase; Thielavia terrestris NRRL 8126 CEL7C
endoglucanase; Thielavia terrestris NRRL 8126 CEL7E endoglucanase;
Thielavia terrestris NRRL 8126 CEL7F endoglucanase; Cladorrhinum
foecundissimum ATCC 62373 CEL7A endoglucanase; and Trichoderma
reesei strain No. VTT-D-80133 endoglucanase; GENBANK.TM. accession
no. M15665.
[0189] Examples of bacterial endoglucanases that can be used in the
methods of the present invention, include, but are not limited to,
an Acidothermus cellulolyticus endoglucanase (WO 91/05039; WO
93/15186; U.S. Pat. No. 5,275,944; WO 96/02551; U.S. Pat. No.
5,536,655, WO 00/70031, WO 05/093050); Thermobifida fusca
endoglucanase III (WO 05/093050); and Thermobifida fusca
endoglucanase V (WO 05/093050).
Cellobiohydrolase I
[0190] The cellulolytic composition used according to the invention
may comprise one or more CBH I (cellobiohydrolase I). The
cellobiohydrolase I may be of any origin, such as microbial origin,
such as eukaryotic origin, such as fungal origin, e.g., filamentous
origin.
[0191] In one embodiment the cellulolytic enzyme composition
comprises a cellobiohydrolase I (CBHI), such as one derived or
isolated from a strain of Aspergillus, such as a strain of
Aspergillus fumigatus, such as the Cel7A CBH I disclosed in SEQ ID
NO: 6 in WO 2011/057140 or SEQ ID NO: 6 herein, or a strain of
Trichoderma, such as a strain of Trichoderma reesei.
[0192] In an embodiment the Aspergillus fumigatus cellobiohydrolase
I (CBH I) or homolog thereof is selected from the group consisting
of:
[0193] (i) a cellobiohydrolase I comprising the mature polypeptide
of SEQ ID NO: 6 herein;
[0194] (ii) a cellobiohydrolase I comprising an amino acid sequence
having at least 70%, e.g., at least 75%, at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99% identity to the mature polypeptide of SEQ ID NO: 6
herein;
[0195] (iii) a cellobiohydrolase I encoded by a polynucleotide
comprising a nucleotide sequence having at least 70%, e.g., at
least 75%, at least 80%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% identity to the
mature polypeptide coding sequence of SEQ ID NO: 5 in WO
2011/057140 (hereby incorporated by reference); and
[0196] (iv) a cellobiohydrolase I encoded by a polynucleotide that
hybridizes under low, medium, high, or very high stringency
conditions, with the mature polypeptide coding sequence of SEQ ID
NO: 5 in WO 2011/057140 or the full-length complement thereof.
Cellobiohydrolase II
[0197] The cellulolytic composition used according to the invention
may comprise one or more CBH II (cellobiohydrolase II). The
cellobiohydrolase II may be of any origin, such as microbial
origin, such as eukaryotic origin, such as fungal origin, e.g.,
filamentous origin.
[0198] In one embodiment the cellobiohydrolase II (CBHII), such as
one derived from a strain of Aspergillus, such as a strain of
Aspergillus fumigatus, such as the one in SEQ ID NO: 7 herein or a
strain of Trichoderma, such as Trichoderma reesei, or a strain of
Thielavia, such as a strain of Thielavia terrestris, such as
cellobiohydrolase II CEL6A from Thielavia terrestris.
[0199] In an embodiment the Aspergillus fumigatus cellobiohydrolase
II or homolog thereof is selected from the group consisting of:
[0200] (i) a cellobiohydrolase II comprising the mature polypeptide
of SEQ ID NO: 4;
[0201] (ii) a cellobiohydrolase II comprising an amino acid
sequence having at least 70%, e.g., at least 75%, at least 80%, at
least 85%, at least 90%, at least 91%, at least 92%, at least 93%,
at least 94%, at least 95%, at least 96%, at least 97%, at least
98%, or at least 99% identity to the mature polypeptide of SEQ ID
NO: 7 herein;
[0202] (iii) a cellobiohydrolase II encoded by a polynucleotide
comprising a nucleotide sequence having at least 70%, e.g., at
least 75%, at least 80%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% identity to the
mature polypeptide coding sequence of SEQ ID NO: 3 in WO
2013/028928 (hereby incorporated by reference); and
[0203] (iv) a cellobiohydrolase II encoded by a polynucleotide that
hybridizes under low, medium, or high stringency conditions, e.g.,
very high stringency conditions, with the mature polypeptide coding
sequence of SEQ ID NO: 3 in WO 2013/028928 or the full-length
complement thereof.
GH61 Polypeptides
[0204] A GH61 polypeptide is according to the invention present
during saccharification together with a cellulolytic enzyme
composition. The GH61 polypeptide may be of any origin, such as
microbial origin, such as eukaryotic origin, such as fungal origin,
e.g., filamentous origin.
[0205] The GH61 polypeptide may be added separately, simultaneously
with or as part of the cellulolytic enzyme composition.
[0206] The GH61 polypeptide may be native or foreign to the strain
from which the cellulolytic enzyme composition is derived or
isolated, such as a strain of Trichoderma reesei, Humicola
insolens, Talaromyces emersonii, or Chrysosporium lucknowense
(Myceliophthora thermophila). In an embodiment the GH61 polypeptide
is a recombinant GH61 polypeptide. In an embodiment the GH61
polypeptide is not of the same origin as the cellulolytic enzyme
composition's host cell, e.g., not of Trichoderma origin, such as
not of Trichoderma reesei origin. In an embodiment the GH61
polypeptide is produced recombinantly as part of the cellulolytic
enzyme composition, e.g., produced by the Trichoderma reesei host
cell producing the cellulolytic enzyme composition.
[0207] In one embodiment the GH61 polypeptide is derived or
isolated from Thermoascus, such as a strain of Thermoascus
aurantiacus, such as the one described in WO 2005/074656 as SEQ ID
NO: 2 and SEQ ID NO: 1 herein; or derived or isolated from
Thielavia, such as a strain of Thielavia terrestris, such as the
one described in WO 2005/074647 as SEQ ID NO: 8 or SEQ ID NO: 4
herein; or derived or isolated from a strain of Aspergillus, such
as a strain of Aspergillus fumigatus, such as the one described in
WO 2010/138754 as SEQ ID NO: 2 or SEQ ID NO: 3 herein; or derived
or isolated from a strain of Penicillium, such as a strain of
Penicillium emersonii, such as the one disclosed as SEQ ID NO: 2 in
WO 2011/041397 or SEQ ID NO: 2 herein.
[0208] In an embodiment the Penicillium sp. GH61 polypeptide or
homolog thereof is selected from the group consisting of:
[0209] (i) a GH61 polypeptide comprising the mature polypeptide of
SEQ ID NO: 8 herein;
[0210] (ii) a GH61 polypeptide comprising an amino acid sequence
having at least 70%, e.g., at least 75%, at least 80%, at least
85%, at least 90%, at least 91%, at least 92%, at least 93%, at
least 94%, at least 95%, at least 96%, at least 97%, at least 98%,
or at least 99% identity to the mature polypeptide of SEQ ID NO: 8
herein;
[0211] (iii) a GH61 polypeptide encoded by a polynucleotide
comprising a nucleotide sequence having at least 70%, e.g., at
least 75%, at least 80%, at least 85%, at least 90%, at least 91%,
at least 92%, at least 93%, at least 94%, at least 95%, at least
96%, at least 97%, at least 98%, or at least 99% identity to the
mature polypeptide coding sequence of SEQ ID NO: 1 in WO
2011/041397 (hereby incorporated by reference); and
[0212] (iv) a GH61 polypeptide encoded by a polynucleotide that
hybridizes under low, medium, high, or very high stringency
conditions, with the mature polypeptide coding sequence of SEQ ID
NO: 1 in WO 2011/041397 or the full-length complement thereof.
[0213] In an embodiment the polypeptide or homolog thereof is
selected from the group consisting of a GH61 polypeptide comprising
the mature polypeptide of SEQ ID NO: 2 in WO 2005/074656; SEQ ID
NO: 8 in WO 2005/074647; SEQ ID NO: 2 in WO 2010/138754; or a GH61
polypeptide comprising an amino acid sequence having at least 70%,
e.g., at least 75%, at least 80%, at least 85%, at least 90%, at
least 91%, at least 92%, at least 93%, at least 94%, at least 95%,
at least 96%, at least 97%, at least 98%, or at least 99% identity
to the mature polypeptide of SEQ ID NO: 2 in WO 2005/074656; SEQ ID
NO: 8 in WO 2005/074647; or SEQ ID NO: 2 in WO 2010/138754 (all
references and sequences hereby incorporated by reference).
[0214] In an embodiment the GH61 polypeptide constitutes from
0.1-25%, such as 0.5-20%, 0.5-15%, 0.5-10%, or 0.5-7% of the
cellulolytic enzyme composition. In an embodiment the amount of
GH61 polypeptide to cellulolytic enzyme composition is about 1 g to
about 1000 g, such as about 1 g to about 200 g, about 1 g to about
100 g, about 1 g to about 50 g, about 1 g to about 20 g, about 1 g
to about 15 g, about 1 g to about 10 g, about 1 g to about 7 g, or
about 1 g to about 4 g per g of cellulolytic enzyme
composition.
Specific Cellulosic Enzyme Compositions Comprising a GH61
Polypeptide
[0215] The following is a list of a number of cellulolytic enzyme
compositions comprising a GH61 polypeptide for use in the present
invention.
[0216] In an embodiment the cellulolytic enzyme composition
comprises a Trichoderma reesei cellulolytic enzyme composition,
further comprising a Thermoascus aurantiacus GH61A polypeptide (WO
2005/074656 and SEQ ID NO: 1 herein) and an Aspergillus oryzae
beta-glucosidase fusion protein (see WO 2008/057637--Examples
10-15).
[0217] In another embodiment the cellulolytic enzyme composition
comprises a Trichoderma reesei cellulolytic enzyme composition,
further comprising a Thermoascus aurantiacus GH61A polypeptide (SEQ
ID NO: 2 in WO 2005/074656 or SEQ ID NO: 1 herein) and an
Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO
2005/047499 or SEQ ID NO: 5 herein).
[0218] In another embodiment the cellulolytic composition comprises
a Trichoderma reesei cellulolytic enzyme composition, further
comprising a Penicillium emersonii GH61A polypeptide disclosed as
SEQ ID NO: 2 in WO 2011/041397 or SEQ ID NO: 2 herein, an
Aspergillus fumigatus beta-glucosidase (SEQ ID NO: 2 of WO
2005/047499 or SEQ ID NO: 5 herein) or a variant thereof with the
following substitutions: F100D, S283G, N456E, F512Y (using SEQ ID
NO: 5 herein for numbering)(disclosed in WO 2012/044915).
Formulation of Cellulolytic Enzyme Compositions
[0219] A cellulolytic enzyme composition used according to the
invention may be in any form suitable for use, such as, for
example, a crude fermentation broth with or without cells removed,
a cell lysate with or without cellular debris, a semi-purified or
purified enzyme composition, or a host cell, e.g., Trichoderma host
cell, as a source of the enzymes.
[0220] The cellulolytic enzyme composition may be a dry powder or
granulate, a non-dusting granulate, a liquid, a stabilized liquid,
or a stabilized protected enzyme. Liquid enzyme compositions may,
for instance, be stabilized by adding stabilizers such as a sugar,
a sugar alcohol or another polyol, and/or lactic acid or another
organic acid according to established processes.
Commercial Cellulolytic Enzyme Compositions
[0221] The cellulolytic enzyme compositions used in accordance with
the methods of the invention may be a commercial cellulolytic
enzyme composition. Examples of commercial cellulolytic enzyme
composition suitable for use according to the present invention
include, for example, CELLIC.TM. CTec (Novozymes A/S), CELLIC.TM.
CTec2 (Novozymes A/S), CELLIC.TM. CTec3 (Novozymes A/S),
CELLUCLAST.TM. (Novozymes A/S), NOVOZYM.TM. 188 (Novozymes NS),
CELLUZYME.TM. (Novozymes A/S), CEREFLO.TM. (Novozymes A/S), and
ULTRAFLO.TM. (Novozymes A/S), ACCELERASE.TM. (DuPont),
ACCELERASE.TM. 1000; ACCELERASE.TM. 1500; ACCELERASE.TM. TRIO;
ACCELERASE.TM. DUET (DuPont); LAMINEX.TM. (Genencor Int.),
SPEZYME.TM. CP (Genencor Int.), ROHAMENT.TM. 7069 W (Rohm GmbH),
FIBREZYME.RTM. LDI (Dyadic International, Inc.), FIBREZYME.RTM. LBR
(Dyadic International, Inc.), or VISCOSTAR.RTM. 150L (Dyadic
International, Inc.). A commercial cellulolytic enzyme composition
may be added in an amount of about 0.001 to about 5.0 wt % of
solids, more preferably from about 0.025 to about 4.0 wt % of
solids, and most preferably from about 0.005 to about 2.0 wt % of
dry solids (DS).
Catalases
[0222] The catalase may be any catalase useful in the processes of
the present invention. The catalase may include, but is not limited
to, an E.C. 1.11.1.6 or E.C. 1.11.1.21 catalase.
[0223] Examples of useful catalases include, but are not limited
to, catalases from Alcaligenes aquamarinus (WO 98/00526),
Aspergillus lentilus, Aspergillus fumigatus, Aspergillus niger
(U.S. Pat. No. 5,360,901), Aspergillus oryzae (JP 2002223772A; U.S.
Pat. No. 6,022,721), Bacillus thermoglucosidasius (JP 1 1243961A),
Humicola insolens (WO 2009/104622, WO 2012/130120), Malbranchea
cinnamomea, Microscilla furvescens (WO 98/00526), Neurospora
crassa, Penicillium emersonii (WO 2012/130120), Penicillium
pinophilum, Rhizomucor pusillus, Saccharomyces pastorianus (WO
2007/105350), Scytalidium thermophilum, Talaromyces stipitatus (WO
2012/130120), Thermoascus aurantiacus (WO 2012/130120), Thermus
brockianus (WO 2005/044994), and Thielavia terrestris (WO
2010/074972).
[0224] Non-limiting examples of catalases useful in the present
invention are catalases from Bacillus pseudofirmus
(UNIPROT:P30266), Bacillus subtilis (UNIPROT: P42234), Humicola
grisea (GeneSeqP: AXQ55105), Neosartorya fischeri (UNIPROT:A1DJU9),
Penicillium emersonii (GeneSeqP:BAC10987), Penicillium pinophilum
(GeneSeqP:BAC10995), Scytalidium thermophilum (GeneSeqP:AAW06109 or
ADT89624), Talaromyces stipitatus (GeneSeqP:BAC10983 or BAC11039;
UNIPROT:B8MT74), and Thermoascus aurantiacus (GeneSeqP:BAC11005).
The accession numbers are incorporated herein in their
entirety.
[0225] In one aspect, the catalase has a sequence identity to the
mature polypeptide of any of the catalases disclosed herein of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%, which have catalase activity.
[0226] In another aspect, the amino acid sequence of the catalase
differs by up to 10 amino acids, e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9,
or 10 from the mature polypeptide of any of the catalases disclosed
herein.
[0227] In another aspect, the catalase comprises or consists of the
amino acid sequence of any of the catalases disclosed herein.
[0228] In another aspect, the catalase comprises or consists of the
mature polypeptide of any of the catalases disclosed herein.
[0229] In another embodiment, the catalase is an allelic variant of
a catalase disclosed herein.
[0230] In another aspect, the catalase is a fragment containing at
least 85% of the amino acid residues, e.g., at least 90% of the
amino acid residues or at least 95% of the amino acid residues of
the mature polypeptide of a catalase disclosed herein.
[0231] In another aspect, the catalase is encoded by a
polynucleotide that hybridizes under very low, low, medium,
medium-high, high, or very high stringency conditions with the
mature polypeptide coding sequence or the full-length complement
thereof of any of the catalases disclosed herein (Sambrook et al.,
1989, supra).
[0232] The polynucleotide encoding a catalase, or a subsequence
thereof, as well as the polypeptide of a catalase, or a fragment
thereof, may be used to design nucleic acid probes to identify and
clone DNA encoding a catalase from strains of different genera or
species according to methods well known in the art. In particular,
such probes can be used for hybridization with the genomic DNA or
cDNA of a cell of interest, as described supra.
[0233] For purposes of the present invention, hybridization
indicates that the polynucleotide hybridizes to a labeled nucleic
acid probe under very low to very high stringency conditions.
Molecules to which the nucleic acid probe hybridizes under these
conditions can be detected using, for example, X-ray film or any
other detection means known in the art.
[0234] In one aspect, the nucleic acid probe is the mature
polypeptide coding sequence of a catalase.
[0235] In another aspect, the nucleic acid probe is a
polynucleotide that encodes a full-length catalase; the mature
polypeptide thereof; or a fragment thereof.
[0236] In another aspect, the catalase is encoded by a
polynucleotide having a sequence identity to the mature polypeptide
coding sequence of any of the catalases disclosed herein of at
least 60%, e.g., at least 65%, at least 70%, at least 75%, at least
80%, at least 81%, at least 82%, at least 83%, at least 84%, at
least 85%, at least 86%, at least 87%, at least 88%, at least 89%,
at least 90%, at least 91%, at least 92%, at least 93%, at least
94%, at least 95%, at least 96%, at least 97%, at least 98%, at
least 99%, or 100%.
[0237] The catalase may be a hybrid polypeptide in which a region
of one polypeptide is fused at the N-terminus or the C-terminus of
a region of another polypeptide or a fusion polypeptide or
cleavable fusion polypeptide in which another polypeptide is fused
at the N-terminus or the C-terminus of the catalase, as described
herein.
[0238] The protein content of the catalase is in the range of about
0.5% to about 10%, e.g., about 0.5% to about 7%, about 0.5% to
about 5%, about 0.5% to about 4%, about 0.5% to about 3%, about
0.5% to about 2%, and about 0.5% to about 1% of total enzyme
protein in the saccharification reaction. In an embodiment, the
protein ratio of catalase to cellulolytic enzyme composition is in
the range of about 1:200 to about 1:10, e.g., about 1:100 to about
1:15 or about 1:50 to about 1:25.
Other Enzymes and Polypeptides Present or Added During
Saccharification
[0239] Other enzymes and/or polypeptides may be present or added
during saccharification. The additional enzymes and/or polypeptide
may be added separately or together with the cellulolytic
composition and/or GH61 polypeptide.
[0240] In an embodiment the cellulolytic enzyme composition
comprises or further comprises one or more (several) enzymes and/or
polypeptides selected from the group consisting of: hemicellulases,
expansins, esterases, laccases, ligninolytic enzymes, pectinases,
peroxidases, proteases, and swollenins.
[0241] In an embodiment the hemicellulase is a xylanase (e.g., an
Aspergillus aculeatus xylanase), an acetyxylan esterase, a feruloyl
esterase, an arabinofuranosidase, a xylosidase, and a
glucuronidase. In a preferred embodiment the hemicellulase is a
xylanase and/or a beta-xylosidase.
[0242] In an embodiment the xylanase is a GH10 xylanase. In an
embodiment the xylanase is derived from a strain of Aspergillus,
such as a strain of Aspergillus fumigatus, such as the one
disclosed as Xyl III in WO 2006/078256 or SEQ ID NO: 9 herein, or
Aspergillus aculeatus, such as the one disclosed in WO 94/21785,
e.g., as Xyl II or SEQ ID NO: 8 herein.
[0243] In an embodiment the beta-xylosidase is derived from a
strain of Aspergillus, such as a strain of Aspergillus fumigatus,
such as the one disclosed in Examples 16-17 as SEQ ID NO: 16 in WO
2013/028928 (hereby incorporated by reference) or SEQ ID NO: 11
herein, or derived from a strain of Trichoderma, such as a strain
of Trichoderma reesei, such as the mature polypeptide of SEQ ID NO:
58 in WO 2011/057140 or SEQ ID NO: 12 herein.
Materials & Methods
Materials:
[0244] CELLUCLAST.TM. 1.5L: Trichoderma reesei cellulolytic enzyme
composition available from Novozymes A/S. GH61 Polypeptide A:
Thermoascus aurantiacus GH61A polypeptide is disclosed in WO
2005/074656 as SEQ ID NO: 2 and is SEQ ID NO: 1 herein. GH61
Polypeptide B: Penicillium sp. (emersonii) GH61 polypeptide is
disclosed in WO 2011/041397 as SEQ ID NO: 2 and is SEQ ID NO: 2
herein. Aspergillus aculeatus beta-glucosidase: obtained according
to Kawaguchi et al., 1996, Gene 173: 287-288. Aspergillus fumigatus
beta-glucosidase (SEQ ID NO: 5 herein) variant is disclosed in WO
2012/044915 with the following substitutions: F100D, S283G, N456E,
F512Y (using SEQ ID NO: 5 for numbering). Aspergillus fumigatus
Cel7A cellobiohydrolase I is disclosed in WO 2011/057140 and is SEQ
ID NO: 6 herein. Aspergillus fumigatus cellobiohydrolase II is
disclosed in WO 2011/057140 and is SEQ ID NO: 7 herein. Aspergillus
fumigatus GH10 xylanase is disclosed in WO 2006/078256 and is SEQ
ID NO: 9 herein. Aspergillus aculeatus xylanase: GH10 xylanase
disclosed in WO 94/21785 as Xyl II (available from Novozymes A/S)
and disclosed as SEQ ID NO: 8 herein. Aspergillus fumigatus
beta-xylosidase is disclosed in WO 2011/057140 and is SEQ ID NO: 11
herein. Cellulolytic Enzyme Composition A: Trichoderma reesei
cellulolytic enzyme composition comprising an Aspergillus fumigatus
Cel7A cellobiohydrolase I (WO 2011/057140), an Aspergillus
fumigatus cellobiohydrolase II (WO 2011/057140), an Aspergillus
fumigatus beta-glucosidase variant disclosed in WO 2012/044915 with
the following substitutions F100D, S283G, N456E, and F512Y, a
Penicillium sp. (emersonii) GH61 polypeptide (WO 2011/041397), an
Aspergillus fumigatus GH10 xylanase (WO 2006/078256), and an
Aspergillus fumigatus beta-xylosidase (WO 2011/057140). Pretreated
Corn Stover (PCS) was supplied by the National Renewable Energy
Laboratory (NREL) in Golden, Colo.
[0245] The present invention is further described by the following
examples that should not be construed as limiting the scope of the
invention.
EXAMPLES
Example 1
Effect of Oxygen During Saccharification with/without GH61
Polypeptide a from Thermoascus aurantiacus
[0246] In order to investigate the effect of dissolved oxygen (DO)
on the yield of dissolved carbohydrates (mainly glucose) when
saccharifying a cellulosic material with a cellulolytic enzyme
composition and a GH61 (family) polypeptide, the following
procedure was used.
Procedure:
[0247] Batches of a pretreated cellulosic material were
saccharified with a cellulolytic enzyme composition with and
without GH61 polypeptide in closed laboratory scale reactors with
continuous agitation, where temperature, pH and dissolved oxygen
(DO) saturation are controlled. The filling volume of the reactors
was 1500 grams of cellulolic material slurry, and the headspace
volume was approximately 1 L. By measuring the final concentration
of dissolved carbohydrates for reactors run with different DO
concentrations, the effect of DO on the yield can be determined for
a cellulolytic enzyme composition with and without a GH61
polypeptide, respectively. Saccharification was run for five days,
the temperature was maintained at 50.degree. C., and the pH was 5.0
for all reactors. The targeted levels of DO were set to values in
the range between 0% and 3% of the saturation level. DO was
maintained by having a controlled flow of nitrogen and air through
the reactor headspace, and good oxygen diffusion between the
headspace and the cellulosic material. Suitable flow rates for air
and nitrogen were in the range 1-100 ml/minute. Good oxygen
diffusion was ensured by running the agitator at high speed (e.g.,
175 rpm). The nitrogen flow was set manually to a constant value,
and the air flow was regulated automatically on the basis of
feedback from a DO sensor (InPro 6800 from Ingold).
Experiment
[0248] The cellulosic material used was corn stover pretreated by
steam explosion in the presence of dilute sulfuric acid. The
pretreated cellulosic material was supplied by the National
Renewable Energy Laboratory (NREL) in Golden, Colo. Total solid
content was 20% and the sulfuric acid concentration was
approximately 0.8%. The cellulosic material was heated to
180.degree. C. for approximately 5 minutes before being discharged
and steam exploded. The pH of the pretreated substrate was adjusted
to 5.0 with sodium hydroxide before enzymes were added.
[0249] The cellulolytic enzyme composition without GH61 polypeptide
consisted of 90% enzyme protein (EP) from CELLUCLAST.TM. 1.5L
(Novozymes A/S), 5% EP from an Aspergillus aculeatus
beta-glucosidase, and 5% EP from Aspergillus acuteatus xylanase. A
dose of 10 mg EP per gram of cellulose was applied.
[0250] The cellulolytic enzyme composition with GH61 polypeptide
consisted of 85% EP from CELLUCLAST.TM. 1.5L, 5% EP of an
Aspergillus aculeatus beta-glucosidase, 5% EP of an Aspergillus
aculeatus xylanase, and 5% EP of a Thermoascus aurantiacus GH61A
polypeptide (GH61 polypeptide A). A dose of 7 mg EP per gram of
cellulose was applied.
[0251] Final dissolved carbohydrate concentrations were measured by
HPLC using an Aminex.RTM. HPX-87H column according to the procedure
described in NREL/TP-510-42623, January 2008. Samples for HPLC were
prepared by centrifuging about 10 g of slurry, transferring 300
microliters of the supernatant to a tube with 10 microliters of 40%
sulfuric acid and 2.09 ml de-ionized water (8.times. dilution), and
filtering through a 0.2 .mu.m syringe filter (Whatman GD/X PTFE, 25
mm diameter).
Results:
[0252] The results of the experiment are shown in FIG. 1. For the
cellulolytic enzyme composition without GH61 polypeptide, the
dissolved oxygen (DO) concentration had little or no effect on the
final dissolved carbohydrate concentration. For the cellulolytic
enzyme composition with GH61 polypeptide, the dissolved oxygen (DO)
concentration had a strong effect on the carbohydrate yield, with
an optimum concentration in the range of 1-2% of the saturation
level and a steep decline below the optimum.
Example 2
Effect of Oxygen Concentration on GH61 Polypeptide B from
Penicillium sp. (emersonii) during Saccharification
[0253] The experiment described in Example 1 was repeated with
Cellulolytic Enzyme Composition A in place of the cellulolytic
enzyme composition described in Example 1. Cellulolytic Enzyme
Composition A was applied at a dose of 5 mg EP per gram of
cellulose.
Results:
[0254] The results shown in FIG. 2 demonstrate that dissolved
oxygen (DO) had a strong effect on the carbohydrate yield, and the
trend was similar to what was observed for the cellulolytic enzyme
composition with GH61 polypeptide described in Example 1.
Example 3
Effect of Dissolved Oxygen and Catalase on the Consumption of
Titrant
[0255] In order to investigate the influence of dissolved oxygen
and catalase addition on sugar yield and consumption of base
titrant, the following experiment was conducted.
[0256] The reactor vessels were IKA LR-2.ST reactor systems with
anchor stirrers. The vessels were each fitted with one Mettler
Toledo InPro 4260 pH and temperature sensor, and one Mettler Toledo
InPro 6800 dissolved oxygen and temperature sensor. The sensors
were connected to a Mettler Toledo M300 transmitter. The sensor
signals were transmitted to a PC. pH was controlled automatically
by dosing a 25% (w/w) solution of sodium hydroxide by a peristaltic
pump controlled by the PC. Air and nitrogen were introduced to the
headspace of the reactors at a rate between 0 and 100 ml/min. The
gas flows were regulated by gas mass flow controllers from
Cole-Parmer (model#32907-59). The flow of nitrogen was controlled
manually, and the flow of air was controlled automatically by the
PC on the basis of the dissolved oxygen sensor signal. The
following three reactors were run:
[0257] (1) Reactor 1. Cellulolytic Enzyme Composition A only with
nitrogen blanketing to purge oxygen from the reactor.
[0258] (2) Reactor 2. Cellulolytic Enzyme Composition A only with
dissolved oxygen (DO) adjustment set to 2% saturation.
[0259] (3) Reactor 3. Cellulolytic Enzyme Composition A and
catalase with DO adjustment set to 2% saturation.
[0260] For each of the reactors above, the substrate was steam
exploded wheat straw with a dry matter content of 20%. The
temperature was set to 50-51.degree. C. with an agitator speed at
75 rpm.
[0261] A total of 830 g liquor and 394 ml water were loaded into
each reactor. The pH of the liquids was adjusted to 5.2-5.3 with
25% NaOH. The solids were added through a funnel in the top of the
reactor under agitation. The pH was maintained at 5.0 for the
remainder of the reaction.
[0262] The enzyme dose for reactor 1 and reactor 2 was Cellulolytic
Enzyme Composition A at 9 mg enzyme protein per g cellulose. The
enzyme dose for reactor 3 was Cellulolytic Enzyme Composition A at
8.78 mg enzyme protein per g cellulose plus 0.217 mg/g
catalase.
[0263] The nitrogen flow was 100 ml/minute initially for all
reactors until the dissolved oxygen concentration was below 10% of
the saturation level. A nitrogen flow of 10 ml/minute to the
headspace was maintained during the trial, in order to protect
against exceeding the DO concentration. The dissolved oxygen
concentration for reactors 2 and 3 was maintained at 2% of the
saturation level for the remainder of the reaction.
[0264] Preparation of samples for HPLC: Slurry samples were removed
from each of the reactor vessels at 3 and 5 days with a serological
pipette and transferred to 15 ml plastic tubes. The samples were
centrifuged for 20 minutes at 4000 rpm in a benchtop centrifuge to
separate liquids and solids. A total of 300 microliters of
supernatant from each sample was diluted with 2.09 ml of DI water
and 6 microliters of 40% H.sub.2SO.sub.4 (8.times. dilution). The
diluted samples were filtered through 0.45 .mu.m PES syringe
filters.
[0265] The sugar concentrations of the samples were measured using
a 300.times.7.7 mm HyperREZ XP Carbohydrate H.sup.+ 8 .mu.m column
equipped with a 0.2 .mu.m inline filter, in place of a guard
column, by elution with 5 mM H.sub.2SO.sub.4 as mobile phase at
65.degree. C. at a flow rate of 0.9 ml per minute, and quantitation
by integration of the glucose signal from refractive index
detection at 50.degree. C. (AGILENT.RTM. 1200 HPLC, Agilent
Technologies, Santa Clara, Calif., USA) calibrated by sugar
standards. The injection volume was 10 microliters and the run time
was 18 minutes.
[0266] The sugar standard was prepared by adding 8.0000 g of
cellobiose, 15.0000 g of glucose, 15.0000 g of xylose, 8.0000 g of
arabinose, 8.0000 g of xylitol, 8.0000 g of glycerol. 12.0000 g of
sodium acetate, and 15.0000 g of ethanol to a 200 mL class A
volumetric flask with 5 mM H.sub.2SO.sub.4 to make a stock solution
(SS). The following dilutions were made in 5 mM H.sub.2SO.sub.4
from the stock solution (SS): [0267] Standard 5=SS [0268] Standard
4=SS diluted 2.times. [0269] Standard 3=SS diluted 4.times. [0270]
Standard 2=SS diluted 20.times. [0271] Standard 1=SS diluted
50.times. [0272] Check Standard=SS diluted 10.times.
[0273] The results are provided in the following tables:
TABLE-US-00001 Amount Glucose Produced (g/) Reactor 1 Reactor 2
Reactor 3 3 days 5 days 3 days 5 days 3 days 5 days 45.2 49.5 61.4
63.3 60.2 67.8
TABLE-US-00002 Amount Xylose Produced (g/) Reactor 1 Reactor 2
Reactor 3 3 days 5 days 3 days 5 days 3 days 5 days 23.3 23.6 24.9
25.1 24.9 26.3
[0274] The table below shows the amount of base consumed during
saccharification to maintain a pH of 5.2 during the reaction.
TABLE-US-00003 Reactor NaOH (25%, ml) 4: Nitrogen 4.9 5: 2% DO 9.6
6: catalase + 2% DO 5.7
[0275] The results show that a low level of dissolved oxygen (2% of
the saturation level) was advantageous for the saccharification
process, as compared to anoxic conditions, but that it led to a
higher consumption (almost doubling) of base titrant required for
maintaining the pH value at the set-point of 5 during the
hydrolysis. The data also demonstrates that the addition of
catalase together with 2% dissolved oxygen saturation significantly
reduced the increase in consumption of titrant and substantially
improved the yield of both xylose and glucose after 5 days of
incubation.
Example 4
Effect of Aeration for a Portion of Hydrolysis
[0276] This experiment was carried out to determine whether or not
aeration is needed for the entire hydrolysis of wheat straw.
[0277] Wheat straw was pretreated by cooking in a two-stage
process. In the first stage cook, the temperature was maintained at
158.degree. C. for 65 minutes, and the liquid was squeezed from the
material after the first stage cook. In the second stage cook, the
squeezed material (dry solids) was subjected to a temperature of
195.degree. C. for 4 minutes. The liquid that was squeezed out
after the first cook and the solids from the second cook were
combined to form pretreated wheat straw.
[0278] Batches of the pretreated wheat straw were hydrolyzed with
Cellulolytic Enzyme Composition A in closed laboratory scale
reactors with continuous agitation, where temperature, pH and
dissolved oxygen (DO) saturation were controlled. The reactor type
was Labfors-5 with 2 L working volume, fitted with helix impellers.
The vessels were each fitted with one Mettler Toledo
405-DPAS-SC-K8S pH sensor and one Mettler Toledo InPro 6820
dissolved oxygen sensor. Flow of air and helium directly into the
hydrolysate was controlled by on/off-valves for the respective
gasses and rotameters incorporated in the control unit, all
supplied by Infors. The flow rates of the gasses were controlled by
the rotameters to 0.4 L/min when the gas flow was switched on. The
pH titrant was 1 M KOH.
[0279] The procedure for loading the pretreated wheat straw into
the reactors was as follows. The pretreated wheat straw and water
were loaded into the reactor. All of the oxygen was removed from
the slurry by flushing with helium. The slurry was heated to
50.degree. C., and the pH was adjusted to 5.3 by manually
controlling the titrant feeding pump. More water was added to reach
a final consistency of 17% total solids. The filling volume of the
reactors was 1200 grams of slurry. The enzyme preparation was added
to the slurry in the reactor. The helium flow was continued until
the pretreated wheat straw was liquefied. Then, the DO control was
switched over to the procedure described below for the given
reactor.
[0280] Saccharification was conducted for five days. The
temperature was maintained at 50.degree. C., and the pH was
maintained at 5.3. The agitator speed was set to 50 RPM.
Cellulolytic Enzyme Composition A was added at a dose of 10 mg EP
per gram. Oxygen was introduced into the reactor at a level of 2%
of the saturation level according to the DO control scheme below.
For the part of hydrolysis in which the oxygen was not introduced,
a controlled flow of helium was added to the reactors. [0281]
Reactor A: DO was introduced at 2% of the saturation level for 24
hours, then helium for the remainder of the saccharification.
[0282] Reactor B: helium for 18 hours, then DO was introduced at 2%
of the saturation level for 24 hours, then helium again for the
remainder of the saccharification. [0283] Reactor C: helium for 44
hours, then DO was introduced at 2% of the saturation level for the
remainder of the saccharification. [0284] Reactor D: Reference
experiment. DO was introduced at 2% of the saturation level for the
entire saccharification.
[0285] Samples of the whole slurry were taken after 2.5, 18.5, 24,
43.8, 48, 94, and 114.8 hours of hydrolysis. Weight/weight
dilutions of the samples were made by thoroughly mixing 2 g of
slurry with 8 g of purified water. A small aliquot was taken from
this dilution, transferred to an Eppendorf tube, and separated by
centrifugation at 14000 RPM in an Eppendorf 5417C centrifuge. The
supernatant was filtered through a 0.22 .mu.m filter and
transferred to an HPLC vial and analyzed for glucose and xylose on
an Aminex.RTM. HPX-87H column according to the procedure described
in NREL/TP-510-42623, January 2008.
[0286] The measured glucose concentrations are shown in the table
below.
TABLE-US-00004 Amount Glucose Produced (g/l) Hours Reactor A
Reactor B Reactor C Reactor D 2.5 13.3 13.5 13.3 13.5 18.5 33.7
26.7 26.6 33.8 24.0 38.5 34.0 29.6 39.1 43.8 43.6 48.7 35.2 51.6
48.0 45.1 49.8 40.0 54.8 94.0 51.7 55.8 63.8 63.8 114.8 53.6 58.0
66.6 65.5
[0287] The late start of oxygen addition (helium for 44 hours, then
DO) did not show an adverse effect on the final glucose
concentration. The addition of oxygen for a short period, either
during the first 24 hours or between 18-44 hours, resulted in a
reduced final glucose concentration. Hence, it is important to
aerate during late stage hydrolysis, but aeration early in the
hydrolysis process is not always necessary.
[0288] The invention described and claimed herein is not to be
limited in scope by the specific aspects herein disclosed, since
these aspects are intended as illustrations of several aspects of
the invention. Any equivalent aspects are intended to be within the
scope of this invention. Indeed, various modifications of the
invention in addition to those shown and described herein will
become apparent to those skilled in the art from the foregoing
description. Such modifications are also intended to fall within
the scope of the appended claims. In the case of conflict, the
present disclosure including definitions will control.
[0289] The present invention is further described in the following
numbered paragraphs:
1. A method of saccharifying a cellulosic material comprising
subjecting the cellulosic material to a cellulolytic enzyme
composition and a GH61 polypeptide in a vessel, wherein oxygen is
added to the vessel to maintain a concentration of dissolved oxygen
in the range of 0.5 to 10% of the saturation level. 2. A method of
saccharifying a cellulosic material comprising subjecting the
cellulosic material to a cellulolytic enzyme composition, a GH61
polypeptide and a catalase in a vessel, wherein oxygen is added to
the vessel to maintain a concentration of dissolved oxygen in the
range of 0.5 to 10% of the saturation level. 3. A method of
saccharifying a cellulosic material comprising subjecting the
cellulosic material to a cellulolytic enzyme composition and a GH61
polypeptide in a vessel, wherein oxygen is added to the vessel to
maintain a concentration of dissolved oxygen in the range of 0.025
ppm to 0.55 ppm, such as, e.g., 0.05 to 0.165 ppm. 4. A method of
saccharifying a cellulosic material comprising subjecting the
cellulosic material to a cellulolytic enzyme composition, a GH61
polypeptide and a catalase in a vessel, wherein oxygen is added to
the vessel to maintain a concentration of dissolved oxygen in the
range of 0.025 ppm to 0.55 ppm, such as, e.g., 0.05 to 0.165 ppm.
5. The method of any of paragraphs 1-4, wherein the amount of
catalase is in the range of 0.5% to 25%, e.g., 0.5% to 20%, 0.5% to
15%, 0.5% to 10%, 0.5% to 7.5%, 0.5% to 5%, and 0.5% to 4% of total
protein. 6. The method of any of paragraphs 1-5, wherein the
dissolved oxygen concentration during saccharification is in the
range of 0.5-10% of the saturation level, such as 0.5-7%, such as
0.5-5%, such as 0.5-4%, such as 0.5-3%, such as 0.5-2%, such as
1-5%, such as 1-4%, such as 1-3%, such as 1-2%. 7. The method of
any of paragraphs 1-6, wherein the dissolved oxygen concentration
is maintained in the range of 0.5-10% of the saturation level, such
as 0.5-7%, such as 0.5-5%, such as 0.5-4%, such as 0.5-3%, such as
0.5-2%, such as 1-5%, such as 1-4%, such as 1-3%, such as 1-2%
during at least 25%, such as at least 50% or at least 75% of the
saccharification period. 8. The method of any of paragraphs 1-7,
wherein the cellulosic material is selected from the group
consisting of herbaceous material (including energy crops),
agricultural residue, wood (including forestry residue), municipal
solid waste, waste paper, pulp, and paper mill residue. 9. The
method of any of paragraphs 1-8, wherein the cellulosic material is
selected from the group consisting of corn stover, wheat straw,
bagasse, corn cob, switchgrass, corn fiber, rice straw, miscanthus,
arundo, bamboo, orange peel, poplar, pine, aspen, fir, spuce,
willow, and eucalyptus. 10. The method of any of paragraphs 1-9,
wherein the cellulosic material is pretreated, e.g., by chemical
and/or physical pretreatment, such as dilute acid and/or steam
explosion pretreatment. 11. The method of any of paragraphs 1-10,
wherein the cellulosic material is pretreated corn stover (PCS),
such as dilute acid pretreated corn stover. 12. The method of any
of paragraphs 1-11, wherein the cellulosic material is unwashed,
such as unwashed pretreated corn stover (uwPCS). 13. The method of
any of paragraphs 1-12, wherein the saccharification occurs for up
to 200 hours, e.g., about 12 to about 96 hours, about 16 to about
72 hours, or about 24 to about 48 hours, such as for at least 12
hours, e.g., at least 24 hours, 36 hours, 48 hours, 60 hours, or 72
hours. 14. The method of any of paragraphs 1-13, wherein the
addition of oxygen to the vessel begins after 24-48 hours of
saccharification and continues until the end of saccharification.
15. The method of any of paragraphs 1-13, wherein the addition of
oxygen occurs throughout the saccharification. 16. The method any
of paragraphs 1-15, wherein the saccharification is performed at a
temperature in the range of about 25.degree. C. to about 75.degree.
C., e.g., about 30.degree. C. to about 70.degree. C., about
35.degree. C. to about 65.degree. C., about 40.degree. C. to
60.degree. C., about 45.degree. C. to 55.degree. C., or about
50.degree. C. 17. The method of any of paragraphs 1-16, wherein the
saccharification is performed at a pH in the range of about 3.0 to
about 7.0, e.g., 3.5 to 6.5, 4.0 to 6.0, 4.5 to 5.5 or about 5.0.
18. The method of paragraph 17, further comprising adding a base to
maintain the pH in the range of about 3.0 to about 7.0, e.g., 3.5
to 6.5, 4.0 to 6.0, 4.5 to 5.5 or about 5.0 during the
saccharification. 19. The method of paragraph 18, wherein the base
is selected from the group consisting of KOH, NaOH, Ca(OH).sub.2,
and NH.sub.4OH. 20. The method of paragraph 18 or 19, wherein the
base is added in an amount of 25-2,500 mmol base per kg dry
cellulosic material, such as 25-1000 mmol/kg, 25-500 mmol/kg,
25-250 mmol/kg, 50-200 mmol/kg. 21. The method of any of paragraphs
1-20, wherein the dry solids content during saccharification (e.g.,
total solids in the cellulosic material) is less than about 30 wt.
%, 25 wt. %, 20 wt. %, 15 wt. %, 10 wt. %, 7.5 wt. %, 5 wt. %, 2.5
wt. %, 2 wt. %, 1 wt. %, or 0.5 wt. %, such as between 5 and 30 wt.
% or between 10 and 25 wt. %. 22. The method of any of paragraphs
1-21, wherein the cellulolytic enzyme composition is of eukaryotic
origin, such as fungal origin, e.g., filamentous origin. 23. The
method of any of paragraphs 1-22, wherein the cellulolytic enzyme
composition is derived from Trichoderma (e.g., Trichoderma reesei).
24. The method of any of paragraphs 1-23, wherein the cellulolytic
enzyme composition comprises at least a cellobiohydrolase, an
endoglucanase, and a beta-glucosidase. 25. The method of any of
paragraphs 1-24, wherein the cellulolytic enzyme composition
comprises a cellobiohydrolase I, a cellobiohydrolase II, an
endoglucanase, and a beta-glucosidase. 26. The method of any of
paragraphs 1-24, wherein the cellulolytic enzyme composition
comprises a cellobiohydrolase, an endoglucanase, a
beta-glucosidase, and a xylanase. 27. The method of any of
paragraphs 1-24, wherein the cellulolytic enzyme composition
comprises a cellobiohydrolase I, a cellobiohydrolase II, an
endoglucanase, a beta-glucosidase, and a xylanase. 28. The method
of any of paragraphs 1-24, wherein the cellulolytic enzyme
composition comprises a cellobiohydrolase I, a cellobiohydrolase
II, an endoglucanase, a beta-glucosidase, a xylanase, and a
beta-xylosidase. 29. The method of any of paragraphs 24-28, wherein
the cellulolytic enzyme composition further comprises one or more
proteins selected from the group consisting of an acetylmannan
esterase, an acetylxylan esterase, an arabinanase, an
arabinofuranosidase, a CIP, a coumaric acid esterase, an esterase,
an expansin, a feruloyl esterase, a galactosidase, a glucuronidase,
a glucuronoyl esterase, a laccase, a ligninolytic enzyme, a
mannanase, a mannosidase, a pectinase, a peroxidase, a protease,
and a swollenin. 30. The method of any of paragraphs 1-29, wherein
the GH61 polypeptide is derived from Thermoascus, such as a strain
of Thermoascus aurantiacus, such as the one described in WO
2005/074656 as SEQ ID NO: 2 or SEQ ID NO: 1 herein; or derived from
Thielavia, such as a strain of Thielavia terrestris, such as the
one described in WO 2005/074647 as SEQ ID NO: 7 and SEQ ID NO: 8 or
SEQ ID NO: 4 herein; or derived from a strain of Aspergillus, such
as a strain of Aspergillus fumigatus, such as the one described in
WO 2010/138754 as SEQ ID NO: 1 and SEQ ID NO: 2 or SEQ ID NO: 3
herein; or a strain of Penicillium, such as a strain of Penicillium
emersonii, such as the one disclosed in WO 2011/041397 or SEQ ID
NO: 2 herein. 31. The method of any of paragraphs 1-30, wherein the
cellulolytic enzyme composition comprises a beta-glucosidase,
preferably one derived from a strain of Aspergillus, such as
Aspergillus oryzae, such as the one disclosed in WO 02/095014 or
the fusion protein having beta-glucosidase activity disclosed in WO
2008/057637, or Aspergillus fumigatus, such as one disclosed as SEQ
ID NO: 2 in WO 2005/047499 or SEQ ID NO: 5 herein, or an
Aspergillus fumigatus beta-glucosidase variant disclosed in WO
2012/044915; or a strain of Penicillium, such as a strain of
Penicillium brasilianum disclosed as SEQ ID NO: 2 in WO
2007/019442, or a strain of Trichoderma, such as a strain of
Trichoderma reesei. 32. The method of any of paragraphs 1-31,
wherein the cellulosic enzyme composition comprises a xylanase,
preferably a GH10 xylanase, such as one derived from a strain of
Aspergillus, such as a strain of Aspergillus fumigatus, such as the
one disclosed as Xyl III in WO 2006/078256 or SEQ ID NO: 9 herein,
or Aspergillus aculeatus, such as the one disclosed in WO 94/21785
as Xyl II or SEQ ID NO: 8 herein. 33. The method of any of
paragraphs 1-32, wherein the cellulolytic enzyme composition
comprises a beta-xylosidase, such as one derived from a strain of
Aspergillus, such as a strain of Aspergillus fumigatus, such as the
one disclosed in co-pending international application no.
PCT/US2012/052163 or SEQ ID NO: 11 herein, or derived from a strain
of Trichoderma, such as a strain of Trichoderma reesei, such as the
mature polypeptide of SEQ ID NO: 58 in WO 2011/057140 or SEQ ID NO:
12 herein. 34. The method of any of paragraphs 1-33, wherein the
cellulolytic enzyme composition comprises a cellobiohydrolase I
(CBH I), such as one derived from a strain of Aspergillus, such as
a strain of Aspergillus fumigatus, such as the Cel7a CBHI disclosed
in SEQ ID NO: 6 in WO 2011/057140 or SEQ ID NO: 6 herein, or a
strain of Trichoderma, such as a strain of Trichoderma reesei. 35.
The method of any of paragraphs 1-34, wherein the cellulolytic
enzyme composition comprises a cellobiohydrolase II (CBH II), such
as one derived from a strain of Aspergillus, such as a strain of
Aspergillus fumigatus disclosed in SEQ ID NO: 7 herein; or a strain
of Trichoderma, such as Trichoderma reesei, or a strain of
Thielavia, such as a strain of Thielavia terrestris, such as
cellobiohydrolase II CEL6A from Thielavia terrestris. 36. The
method of any of paragraphs 1-35, wherein the cellulolytic enzyme
composition comprises a Trichoderma reesei cellulase composition
and Thermoascus aurantiacus GH61A polypeptide (SEQ ID NO: 2 in WO
2005/074656 or SEQ ID NO: 1 herein). 37. The method of any of
paragraphs 1-36, wherein the cellulolytic enzyme composition
comprises a beta-glucosidase, such as an Aspergillus oryzae
beta-glucosidase fusion protein (WO 2008/057637). 38. The method of
any of paragraphs 1-37, wherein the cellulolytic enzyme composition
is a Trichoderma reesei cellulolytic enzyme composition comprising
a Penicillium emersonii GH61A polypeptide disclosed in WO
2011/041397 or SEQ ID NO: 2 herein. 39. The method of any of
paragraphs 1-38, wherein the cellulolytic enzyme composition
comprises a beta-glucosidase, such as an Aspergillus fumigatus
beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ ID NO: 5
herein), or a variant thereof with the following substitutions:
F100D, S283G, N456E, F512Y (using SEQ ID NO: 5 herein for
numbering). 40. The method of any of paragraphs 1-39, wherein the
cellulolytic enzyme composition is a Trichoderma reesei
cellulolytic enzyme composition comprising one or more of the
following components: [0290] (a) an Aspergillus fumigatus
cellobiohydrolase I; [0291] (b) an Aspergillus fumigatus
cellobiohydrolase II; [0292] (c) an Aspergillus fumigatus
beta-glucosidase or variant thereof with one or more of the
following substitutions: F100D, S283G, N456E, F512Y using SEQ ID
NO: 5 herein for numbering; and [0293] (d) a Penicillium sp. GH61
polypeptide; or homologs thereof. 41. The method of any of
paragraphs 1-40, wherein the cellulolytic enzyme composition is a
Trichoderma reesei cellulolytic enzyme composition, further
comprising a Thermoascus aurantiacus GH61A polypeptide (SEQ ID NO:
1 and SEQ ID NO: 2 in WO 2005/074656 or SEQ ID NO: 1 herein), an
Aspergillus oryzae beta-glucosidase fusion protein (WO
2008/057637), and an Aspergillus aculeatus xylanase (Xyl II in WO
94/21785 or SEQ ID NO: 8 herein). 42. The method of any of
paragraphs 1-41, wherein the cellulolytic enzyme composition is a
Trichoderma reesei cellulolytic enzyme composition, further
comprising a Thermoascus aurantiacus GH61A polypeptide (SEQ ID NO:
2 in WO 2005/074656 or SEQ ID NO: 1 herein), an Aspergillus
fumigatus beta-glucosidase (SEQ ID NO: 2 of WO 2005/047499 or SEQ
ID NO: 5 herein) and an Aspergillus aculeatus xylanase (Xyl II
disclosed in WO 94/21785 or SEQ ID NO: 8 herein). 43. The method of
any of paragraphs 1-42, wherein the cellulolytic enzyme composition
is a Trichoderma reesei cellulolytic enzyme composition, further
comprising a Penicillium emersonii GH61A polypeptide (SEQ ID NO: 2
in WO 2011/04139 or SEQ ID NO: 2 herein), an Aspergillus fumigatus
beta-glucosidase (disclosed as SEQ ID NO: 2 in WO 2005/047499 or
SEQ ID NO: 5 herein), an Aspergillus fumigatus xylanase (Xyl III
disclosed in WO 2006/078256 or SEQ ID NO: 9 herein) and a
beta-xylosidase derived from a strain of Aspergillus fumigatus (SEQ
ID NO: 11 herein). 44. The method of any of paragraphs 1-43,
wherein the cellulolytic enzyme composition is added in an amount
of about 0.01 to about 50.0 mg, e.g., about 1 to about 25 mg, such
as about 2-10 mg, such as about 4 to about 8 mg protein per g/DS of
the cellulosic material. 45. The method of any of paragraphs 1-44,
further comprising recovering the saccharified cellulosic material.
46. The method of paragraph 45, wherein the saccharified cellulosic
material is a sugar. 47. The method of paragraph 46, wherein the
sugar is selected from the group consisting of arabinose,
galactose, glucose, mannose, and xylose. 48. The method of any of
paragraphs 1-47, wherein the GH61 polypeptide constitutes from
0.1-15%, preferably 0.5-10%, and more preferably 0.5-7% of the
cellulolytic enzyme composition. 49. The method of any of
paragraphs 1-48, wherein the vessel comprises more than 10 m.sup.3,
such as more than 25 m.sup.3, such as more than 50 m.sup.3
cellulosic material. 50. A method of producing a fermentation
product from cellulosic material, comprising: [0294] (a)
saccharification of the cellulosic material in accordance with the
method of any of paragraphs 1-49; and [0295] (b) fermenting the
saccharified cellulosic material with one or more fermenting
microorganisms. 51. The method of paragraph 50, further comprising
recovering the fermentation product from (b). 52. The method of
paragraph 50 or 51, wherein the saccharification and fermentation
occur simultaneously or sequentially. 53. The method of any of
paragraphs 50-52, wherein the fermentation occurs for about 8 to
about 96 hours, such as about 24 to about 60 hours. 54. The method
of any of paragraphs 50-53, wherein the fermentation is performed
at a temperature between about 26.degree. C. to about 60.degree.
C., in particular about 32.degree. C. or 50
.degree. C. 55. The method of any of paragraphs 50-54, wherein the
fermentation is performed at about pH 3 to about pH 8, such as
around pH 4-5, 6, or 7. 56. The method of any of paragraphs 50-55,
wherein the fermentation product is an alcohol, an organic acid, a
ketone, an amino acid, or a gas. 57. The method of paragraph 56,
wherein the fermentation product is ethanol. 58. The method of any
of paragraphs 50-57, wherein the fermenting microorganism is a
bacterial or fungal organism. 59. The method of any of paragraphs
50-58, wherein the fermenting organism is a hexose and/or pentose
fermenting organism, or a combination thereof. 60. The method of
any of paragraphs 50-59, wherein the fermenting microorganism is a
strain of the Saccharomyces spp., preferably Saccharomyces
cerevisiae. 61. The method of any of paragraphs 50-60, wherein the
fermenting organism is a strain of Pichia, preferably Pichia
stipitis, such as Pichia stipitis CBS 5773; strain of Candida,
preferably Candida boidinii, Candida brassicae, Candida sheatae,
Candida diddensii, Candida pseudotropicalis, or Candida utilis. 62.
The method of any of paragraphs 50-61, wherein the fermenting
organism is a strain of Zymomonas, such as Zymomonas mobilis;
Hansenula, such as Hansenula anomala; Kluyveromyces, such as K.
marxianus, K. lactis, K. thermotolerans, and K. fragilis;
Schizosaccharomyces, such as S. pombe; E. coli, a strain of
Clostridium, such as Clostridium acetobutylicum, Chlostridium
thermocellum, and Chlostridium phytofermentans; a strain of
Geobacillus sp.; a strain of Thermoanaerobacter, such as
Thermoanaerobacter saccharolyticum; a strain of Bacillus, such as
Bacillus coagulans. 63. The method of any of paragraphs 50-62,
wherein the fermenting microorganism has been genetically modified
to provide the ability to ferment pentose sugars, such as xylose
utilizing, arabinose utilizing, and xylose and arabinose
co-utilizing microorganisms. 64. The method of any of paragraphs
50-63, wherein the fermenting microorganism is a strain of
Saccharomyces spp., such as Saccharomyces cerevisiae, capable of
effectively co-fermenting glucose and xylose. 65. The method of any
of paragraphs 50-64, wherein the fermenting microorganism expresses
xylose isomerase.
Sequence CWU 1
1
111250PRTThermoascus aurantiacus 1Met Ser Phe Ser Lys Ile Ile Ala
Thr Ala Gly Val Leu Ala Ser Ala 1 5 10 15 Ser Leu Val Ala Gly His
Gly Phe Val Gln Asn Ile Val Ile Asp Gly 20 25 30 Lys Lys Tyr Tyr
Gly Gly Tyr Leu Val Asn Gln Tyr Pro Tyr Met Ser 35 40 45 Asn Pro
Pro Glu Val Ile Ala Trp Ser Thr Thr Ala Thr Asp Leu Gly 50 55 60
Phe Val Asp Gly Thr Gly Tyr Gln Thr Pro Asp Ile Ile Cys His Arg 65
70 75 80 Gly Ala Lys Pro Gly Ala Leu Thr Ala Pro Val Ser Pro Gly
Gly Thr 85 90 95 Val Glu Leu Gln Trp Thr Pro Trp Pro Asp Ser His
His Gly Pro Val 100 105 110 Ile Asn Tyr Leu Ala Pro Cys Asn Gly Asp
Cys Ser Thr Val Asp Lys 115 120 125 Thr Gln Leu Glu Phe Phe Lys Ile
Ala Glu Ser Gly Leu Ile Asn Asp 130 135 140 Asp Asn Pro Pro Gly Ile
Trp Ala Ser Asp Asn Leu Ile Ala Ala Asn 145 150 155 160 Asn Ser Trp
Thr Val Thr Ile Pro Thr Thr Ile Ala Pro Gly Asn Tyr 165 170 175 Val
Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Gln Asn Gln Asp 180 185
190 Gly Ala Gln Asn Tyr Pro Gln Cys Ile Asn Leu Gln Val Thr Gly Gly
195 200 205 Gly Ser Asp Asn Pro Ala Gly Thr Leu Gly Thr Ala Leu Tyr
His Asp 210 215 220 Thr Asp Pro Gly Ile Leu Ile Asn Ile Tyr Gln Lys
Leu Ser Ser Tyr 225 230 235 240 Ile Ile Pro Gly Pro Pro Leu Tyr Thr
Gly 245 250 2253PRTPenicillium emersonii 2Met Leu Ser Ser Thr Thr
Arg Thr Leu Ala Phe Thr Gly Leu Ala Gly 1 5 10 15 Leu Leu Ser Ala
Pro Leu Val Lys Ala His Gly Phe Val Gln Gly Ile 20 25 30 Val Ile
Gly Asp Gln Phe Tyr Ser Gly Tyr Ile Val Asn Ser Phe Pro 35 40 45
Tyr Glu Ser Asn Pro Pro Pro Val Ile Gly Trp Ala Thr Thr Ala Thr 50
55 60 Asp Leu Gly Phe Val Asp Gly Thr Gly Tyr Gln Gly Pro Asp Ile
Ile 65 70 75 80 Cys His Arg Asn Ala Thr Pro Ala Pro Leu Thr Ala Pro
Val Ala Ala 85 90 95 Gly Gly Thr Val Glu Leu Gln Trp Thr Pro Trp
Pro Asp Ser His His 100 105 110 Gly Pro Val Ile Thr Tyr Leu Ala Pro
Cys Asn Gly Asn Cys Ser Thr 115 120 125 Val Asp Lys Thr Thr Leu Glu
Phe Phe Lys Ile Asp Gln Gln Gly Leu 130 135 140 Ile Asp Asp Thr Ser
Pro Pro Gly Thr Trp Ala Ser Asp Asn Leu Ile 145 150 155 160 Ala Asn
Asn Asn Ser Trp Thr Val Thr Ile Pro Asn Ser Val Ala Pro 165 170 175
Gly Asn Tyr Val Leu Arg His Glu Ile Ile Ala Leu His Ser Ala Asn 180
185 190 Asn Lys Asp Gly Ala Gln Asn Tyr Pro Gln Cys Ile Asn Ile Glu
Val 195 200 205 Thr Gly Gly Gly Ser Asp Ala Pro Glu Gly Thr Leu Gly
Glu Asp Leu 210 215 220 Tyr His Asp Thr Asp Pro Gly Ile Leu Val Asp
Ile Tyr Glu Pro Ile 225 230 235 240 Ala Thr Tyr Thr Ile Pro Gly Pro
Pro Glu Pro Thr Phe 245 250 3326PRTAspergillus fumigatus 3Met Lys
Ser Phe Thr Ile Ala Ala Leu Ala Ala Leu Trp Ala Gln Glu 1 5 10 15
Ala Ala Ala His Ala Thr Phe Gln Asp Leu Trp Ile Asp Gly Val Asp 20
25 30 Tyr Gly Ser Gln Cys Val Arg Leu Pro Ala Ser Asn Ser Pro Val
Thr 35 40 45 Asn Val Ala Ser Asp Asp Ile Arg Cys Asn Val Gly Thr
Ser Arg Pro 50 55 60 Thr Val Lys Cys Pro Val Lys Ala Gly Ser Thr
Val Thr Ile Glu Met 65 70 75 80 His Gln Gln Pro Gly Asp Arg Ser Cys
Ala Asn Glu Ala Ile Gly Gly 85 90 95 Asp His Tyr Gly Pro Val Met
Val Tyr Met Ser Lys Val Asp Asp Ala 100 105 110 Val Thr Ala Asp Gly
Ser Ser Gly Trp Phe Lys Val Phe Gln Asp Ser 115 120 125 Trp Ala Lys
Asn Pro Ser Gly Ser Thr Gly Asp Asp Asp Tyr Trp Gly 130 135 140 Thr
Lys Asp Leu Asn Ser Cys Cys Gly Lys Met Asn Val Lys Ile Pro 145 150
155 160 Glu Asp Ile Glu Pro Gly Asp Tyr Leu Leu Arg Ala Glu Val Ile
Ala 165 170 175 Leu His Val Ala Ala Ser Ser Gly Gly Ala Gln Phe Tyr
Met Ser Cys 180 185 190 Tyr Gln Leu Thr Val Thr Gly Ser Gly Ser Ala
Thr Pro Ser Thr Val 195 200 205 Asn Phe Pro Gly Ala Tyr Ser Ala Ser
Asp Pro Gly Ile Leu Ile Asn 210 215 220 Ile His Ala Pro Met Ser Thr
Tyr Val Val Pro Gly Pro Thr Val Tyr 225 230 235 240 Ala Gly Gly Ser
Thr Lys Ser Ala Gly Ser Ser Cys Ser Gly Cys Glu 245 250 255 Ala Thr
Cys Thr Val Gly Ser Gly Pro Ser Ala Thr Leu Thr Gln Pro 260 265 270
Thr Ser Thr Ala Thr Ala Thr Ser Ala Pro Gly Gly Gly Gly Ser Gly 275
280 285 Cys Thr Ala Ala Lys Tyr Gln Gln Cys Gly Gly Thr Gly Tyr Thr
Gly 290 295 300 Cys Thr Thr Cys Ala Ser Gly Ser Thr Cys Ser Ala Val
Ser Pro Pro 305 310 315 320 Tyr Tyr Ser Gln Cys Leu 325
4452PRTThielavia terrestris 4Met Leu Ala Asn Gly Ala Ile Val Phe
Leu Ala Ala Ala Leu Gly Val 1 5 10 15 Ser Gly His Tyr Thr Trp Pro
Arg Val Asn Asp Gly Ala Asp Trp Gln 20 25 30 Gln Val Arg Lys Ala
Asp Asn Trp Gln Asp Asn Gly Tyr Val Gly Asp 35 40 45 Val Thr Ser
Pro Gln Ile Arg Cys Phe Gln Ala Thr Pro Ser Pro Ala 50 55 60 Pro
Ser Val Leu Asn Thr Thr Ala Gly Ser Thr Val Thr Tyr Trp Ala 65 70
75 80 Asn Pro Asp Val Tyr His Pro Gly Pro Val Gln Phe Tyr Met Ala
Arg 85 90 95 Val Pro Asp Gly Glu Asp Ile Asn Ser Trp Asn Gly Asp
Gly Ala Val 100 105 110 Trp Phe Lys Val Tyr Glu Asp His Pro Thr Phe
Gly Ala Gln Leu Thr 115 120 125 Trp Pro Ser Thr Gly Lys Ser Ser Phe
Ala Val Pro Ile Pro Pro Cys 130 135 140 Ile Lys Ser Gly Tyr Tyr Leu
Leu Arg Ala Glu Gln Ile Gly Leu His 145 150 155 160 Val Ala Gln Ser
Val Gly Gly Ala Gln Phe Tyr Ile Ser Cys Ala Gln 165 170 175 Leu Ser
Val Thr Gly Gly Gly Ser Thr Glu Pro Pro Asn Lys Val Ala 180 185 190
Phe Pro Gly Ala Tyr Ser Ala Thr Asp Pro Gly Ile Leu Ile Asn Ile 195
200 205 Tyr Tyr Pro Val Pro Thr Ser Tyr Gln Asn Pro Gly Pro Ala Val
Phe 210 215 220 Ser Cys Met Leu Ala Asn Gly Ala Ile Val Phe Leu Ala
Ala Ala Leu 225 230 235 240 Gly Val Ser Gly His Tyr Thr Trp Pro Arg
Val Asn Asp Gly Ala Asp 245 250 255 Trp Gln Gln Val Arg Lys Ala Asp
Asn Trp Gln Asp Asn Gly Tyr Val 260 265 270 Gly Asp Val Thr Ser Pro
Gln Ile Arg Cys Phe Gln Ala Thr Pro Ser 275 280 285 Pro Ala Pro Ser
Val Leu Asn Thr Thr Ala Gly Ser Thr Val Thr Tyr 290 295 300 Trp Ala
Asn Pro Asp Val Tyr His Pro Gly Pro Val Gln Phe Tyr Met 305 310 315
320 Ala Arg Val Pro Asp Gly Glu Asp Ile Asn Ser Trp Asn Gly Asp Gly
325 330 335 Ala Val Trp Phe Lys Val Tyr Glu Asp His Pro Thr Phe Gly
Ala Gln 340 345 350 Leu Thr Trp Pro Ser Thr Gly Lys Ser Ser Phe Ala
Val Pro Ile Pro 355 360 365 Pro Cys Ile Lys Ser Gly Tyr Tyr Leu Leu
Arg Ala Glu Gln Ile Gly 370 375 380 Leu His Val Ala Gln Ser Val Gly
Gly Ala Gln Phe Tyr Ile Ser Cys 385 390 395 400 Ala Gln Leu Ser Val
Thr Gly Gly Gly Ser Thr Glu Pro Pro Asn Lys 405 410 415 Val Ala Phe
Pro Gly Ala Tyr Ser Ala Thr Asp Pro Gly Ile Leu Ile 420 425 430 Asn
Ile Tyr Tyr Pro Val Pro Thr Ser Tyr Gln Asn Pro Gly Pro Ala 435 440
445 Val Phe Ser Cys 450 5863PRTAspergillus fumigatus 5Met Arg Phe
Gly Trp Leu Glu Val Ala Ala Leu Thr Ala Ala Ser Val 1 5 10 15 Ala
Asn Ala Gln Glu Leu Ala Phe Ser Pro Pro Phe Tyr Pro Ser Pro 20 25
30 Trp Ala Asp Gly Gln Gly Glu Trp Ala Asp Ala His Arg Arg Ala Val
35 40 45 Glu Ile Val Ser Gln Met Thr Leu Ala Glu Lys Val Asn Leu
Thr Thr 50 55 60 Gly Thr Gly Trp Glu Met Asp Arg Cys Val Gly Gln
Thr Gly Ser Val 65 70 75 80 Pro Arg Leu Gly Ile Asn Trp Gly Leu Cys
Gly Gln Asp Ser Pro Leu 85 90 95 Gly Ile Arg Phe Ser Asp Leu Asn
Ser Ala Phe Pro Ala Gly Thr Asn 100 105 110 Val Ala Ala Thr Trp Asp
Lys Thr Leu Ala Tyr Leu Arg Gly Lys Ala 115 120 125 Met Gly Glu Glu
Phe Asn Asp Lys Gly Val Asp Ile Leu Leu Gly Pro 130 135 140 Ala Ala
Gly Pro Leu Gly Lys Tyr Pro Asp Gly Gly Arg Ile Trp Glu 145 150 155
160 Gly Phe Ser Pro Asp Pro Val Leu Thr Gly Val Leu Phe Ala Glu Thr
165 170 175 Ile Lys Gly Ile Gln Asp Ala Gly Val Ile Ala Thr Ala Lys
His Tyr 180 185 190 Ile Leu Asn Glu Gln Glu His Phe Arg Gln Val Gly
Glu Ala Gln Gly 195 200 205 Tyr Gly Tyr Asn Ile Thr Glu Thr Ile Ser
Ser Asn Val Asp Asp Lys 210 215 220 Thr Met His Glu Leu Tyr Leu Trp
Pro Phe Ala Asp Ala Val Arg Ala 225 230 235 240 Gly Val Gly Ala Val
Met Cys Ser Tyr Asn Gln Ile Asn Asn Ser Tyr 245 250 255 Gly Cys Gln
Asn Ser Gln Thr Leu Asn Lys Leu Leu Lys Ala Glu Leu 260 265 270 Gly
Phe Gln Gly Phe Val Met Ser Asp Trp Ser Ala His His Ser Gly 275 280
285 Val Gly Ala Ala Leu Ala Gly Leu Asp Met Ser Met Pro Gly Asp Ile
290 295 300 Ser Phe Asp Asp Gly Leu Ser Phe Trp Gly Thr Asn Leu Thr
Val Ser 305 310 315 320 Val Leu Asn Gly Thr Val Pro Ala Trp Arg Val
Asp Asp Met Ala Val 325 330 335 Arg Ile Met Thr Ala Tyr Tyr Lys Val
Gly Arg Asp Arg Leu Arg Ile 340 345 350 Pro Pro Asn Phe Ser Ser Trp
Thr Arg Asp Glu Tyr Gly Trp Glu His 355 360 365 Ser Ala Val Ser Glu
Gly Ala Trp Thr Lys Val Asn Asp Phe Val Asn 370 375 380 Val Gln Arg
Ser His Ser Gln Ile Ile Arg Glu Ile Gly Ala Ala Ser 385 390 395 400
Thr Val Leu Leu Lys Asn Thr Gly Ala Leu Pro Leu Thr Gly Lys Glu 405
410 415 Val Lys Val Gly Val Leu Gly Glu Asp Ala Gly Ser Asn Pro Trp
Gly 420 425 430 Ala Asn Gly Cys Pro Asp Arg Gly Cys Asp Asn Gly Thr
Leu Ala Met 435 440 445 Ala Trp Gly Ser Gly Thr Ala Asn Phe Pro Tyr
Leu Val Thr Pro Glu 450 455 460 Gln Ala Ile Gln Arg Glu Val Ile Ser
Asn Gly Gly Asn Val Phe Ala 465 470 475 480 Val Thr Asp Asn Gly Ala
Leu Ser Gln Met Ala Asp Val Ala Ser Gln 485 490 495 Ser Ser Val Ser
Leu Val Phe Val Asn Ala Asp Ser Gly Glu Gly Phe 500 505 510 Ile Ser
Val Asp Gly Asn Glu Gly Asp Arg Lys Asn Leu Thr Leu Trp 515 520 525
Lys Asn Gly Glu Ala Val Ile Asp Thr Val Val Ser His Cys Asn Asn 530
535 540 Thr Ile Val Val Ile His Ser Val Gly Pro Val Leu Ile Asp Arg
Trp 545 550 555 560 Tyr Asp Asn Pro Asn Val Thr Ala Ile Ile Trp Ala
Gly Leu Pro Gly 565 570 575 Gln Glu Ser Gly Asn Ser Leu Val Asp Val
Leu Tyr Gly Arg Val Asn 580 585 590 Pro Ser Ala Lys Thr Pro Phe Thr
Trp Gly Lys Thr Arg Glu Ser Tyr 595 600 605 Gly Ala Pro Leu Leu Thr
Glu Pro Asn Asn Gly Asn Gly Ala Pro Gln 610 615 620 Asp Asp Phe Asn
Glu Gly Val Phe Ile Asp Tyr Arg His Phe Asp Lys 625 630 635 640 Arg
Asn Glu Thr Pro Ile Tyr Glu Phe Gly His Gly Leu Ser Tyr Thr 645 650
655 Thr Phe Gly Tyr Ser His Leu Arg Val Gln Ala Leu Asn Ser Ser Ser
660 665 670 Ser Ala Tyr Val Pro Thr Ser Gly Glu Thr Lys Pro Ala Pro
Thr Tyr 675 680 685 Gly Glu Ile Gly Ser Ala Ala Asp Tyr Leu Tyr Pro
Glu Gly Leu Lys 690 695 700 Arg Ile Thr Lys Phe Ile Tyr Pro Trp Leu
Asn Ser Thr Asp Leu Glu 705 710 715 720 Asp Ser Ser Asp Asp Pro Asn
Tyr Gly Trp Glu Asp Ser Glu Tyr Ile 725 730 735 Pro Glu Gly Ala Arg
Asp Gly Ser Pro Gln Pro Leu Leu Lys Ala Gly 740 745 750 Gly Ala Pro
Gly Gly Asn Pro Thr Leu Tyr Gln Asp Leu Val Arg Val 755 760 765 Ser
Ala Thr Ile Thr Asn Thr Gly Asn Val Ala Gly Tyr Glu Val Pro 770 775
780 Gln Leu Tyr Val Ser Leu Gly Gly Pro Asn Glu Pro Arg Val Val Leu
785 790 795 800 Arg Lys Phe Asp Arg Ile Phe Leu Ala Pro Gly Glu Gln
Lys Val Trp 805 810 815 Thr Thr Thr Leu Asn Arg Arg Asp Leu Ala Asn
Trp Asp Val Glu Ala 820 825 830 Gln Asp Trp Val Ile Thr Lys Tyr Pro
Lys Lys Val His Val Gly Ser 835 840 845 Ser Ser Arg Lys Leu Pro Leu
Arg Ala Pro Leu Pro Arg Val Tyr 850 855 860 6532PRTAspergillus
fumigatus 6Met Leu Ala Ser Thr Phe Ser Tyr Arg Met Tyr Lys Thr Ala
Leu Ile 1 5 10 15 Leu Ala Ala Leu Leu Gly Ser Gly Gln Ala Gln Gln
Val Gly Thr Ser 20 25 30 Gln Ala Glu Val His Pro Ser Met Thr Trp
Gln Ser Cys Thr Ala Gly 35 40 45 Gly Ser Cys Thr Thr Asn Asn Gly
Lys Val Val Ile Asp Ala Asn Trp 50 55 60 Arg Trp Val His Lys Val
Gly Asp Tyr Thr Asn Cys Tyr Thr Gly Asn 65 70 75 80 Thr Trp Asp Thr
Thr Ile Cys Pro Asp Asp Ala Thr Cys Ala Ser Asn 85 90 95 Cys Ala
Leu Glu Gly Ala Asn Tyr Glu Ser Thr Tyr Gly Val Thr Ala 100 105 110
Ser Gly Asn Ser Leu Arg Leu Asn Phe Val Thr Thr Ser Gln Gln Lys 115
120 125 Asn Ile Gly Ser Arg Leu Tyr Met Met Lys Asp Asp Ser Thr Tyr
Glu 130 135
140 Met Phe Lys Leu Leu Asn Gln Glu Phe Thr Phe Asp Val Asp Val Ser
145 150 155 160 Asn Leu Pro Cys Gly Leu Asn Gly Ala Leu Tyr Phe Val
Ala Met Asp 165 170 175 Ala Asp Gly Gly Met Ser Lys Tyr Pro Thr Asn
Lys Ala Gly Ala Lys 180 185 190 Tyr Gly Thr Gly Tyr Cys Asp Ser Gln
Cys Pro Arg Asp Leu Lys Phe 195 200 205 Ile Asn Gly Gln Ala Asn Val
Glu Gly Trp Gln Pro Ser Ser Asn Asp 210 215 220 Ala Asn Ala Gly Thr
Gly Asn His Gly Ser Cys Cys Ala Glu Met Asp 225 230 235 240 Ile Trp
Glu Ala Asn Ser Ile Ser Thr Ala Phe Thr Pro His Pro Cys 245 250 255
Asp Thr Pro Gly Gln Val Met Cys Thr Gly Asp Ala Cys Gly Gly Thr 260
265 270 Tyr Ser Ser Asp Arg Tyr Gly Gly Thr Cys Asp Pro Asp Gly Cys
Asp 275 280 285 Phe Asn Ser Phe Arg Gln Gly Asn Lys Thr Phe Tyr Gly
Pro Gly Met 290 295 300 Thr Val Asp Thr Lys Ser Lys Phe Thr Val Val
Thr Gln Phe Ile Thr 305 310 315 320 Asp Asp Gly Thr Ser Ser Gly Thr
Leu Lys Glu Ile Lys Arg Phe Tyr 325 330 335 Val Gln Asn Gly Lys Val
Ile Pro Asn Ser Glu Ser Thr Trp Thr Gly 340 345 350 Val Ser Gly Asn
Ser Ile Thr Thr Glu Tyr Cys Thr Ala Gln Lys Ser 355 360 365 Leu Phe
Gln Asp Gln Asn Val Phe Glu Lys His Gly Gly Leu Glu Gly 370 375 380
Met Gly Ala Ala Leu Ala Gln Gly Met Val Leu Val Met Ser Leu Trp 385
390 395 400 Asp Asp His Ser Ala Asn Met Leu Trp Leu Asp Ser Asn Tyr
Pro Thr 405 410 415 Thr Ala Ser Ser Thr Thr Pro Gly Val Ala Arg Gly
Thr Cys Asp Ile 420 425 430 Ser Ser Gly Val Pro Ala Asp Val Glu Ala
Asn His Pro Asp Ala Tyr 435 440 445 Val Val Tyr Ser Asn Ile Lys Val
Gly Pro Ile Gly Ser Thr Phe Asn 450 455 460 Ser Gly Gly Ser Asn Pro
Gly Gly Gly Thr Thr Thr Thr Thr Thr Thr 465 470 475 480 Gln Pro Thr
Thr Thr Thr Thr Thr Ala Gly Asn Pro Gly Gly Thr Gly 485 490 495 Val
Ala Gln His Tyr Gly Gln Cys Gly Gly Ile Gly Trp Thr Gly Pro 500 505
510 Thr Thr Cys Ala Ser Pro Tyr Thr Cys Gln Lys Leu Asn Asp Tyr Tyr
515 520 525 Ser Gln Cys Leu 530 7454PRTAspergillus fumigatus 7Met
Lys His Leu Ala Ser Ser Ile Ala Leu Thr Leu Leu Leu Pro Ala 1 5 10
15 Val Gln Ala Gln Gln Thr Val Trp Gly Gln Cys Gly Gly Gln Gly Trp
20 25 30 Ser Gly Pro Thr Ser Cys Val Ala Gly Ala Ala Cys Ser Thr
Leu Asn 35 40 45 Pro Tyr Tyr Ala Gln Cys Ile Pro Gly Ala Thr Ala
Thr Ser Thr Thr 50 55 60 Leu Thr Thr Thr Thr Ala Ala Thr Thr Thr
Ser Gln Thr Thr Thr Lys 65 70 75 80 Pro Thr Thr Thr Gly Pro Thr Thr
Ser Ala Pro Thr Val Thr Ala Ser 85 90 95 Gly Asn Pro Phe Ser Gly
Tyr Gln Leu Tyr Ala Asn Pro Tyr Tyr Ser 100 105 110 Ser Glu Val His
Thr Leu Ala Met Pro Ser Leu Pro Ser Ser Leu Gln 115 120 125 Pro Lys
Ala Ser Ala Val Ala Glu Val Pro Ser Phe Val Trp Leu Asp 130 135 140
Val Ala Ala Lys Val Pro Thr Met Gly Thr Tyr Leu Ala Asp Ile Gln 145
150 155 160 Ala Lys Asn Lys Ala Gly Ala Asn Pro Pro Ile Ala Gly Ile
Phe Val 165 170 175 Val Tyr Asp Leu Pro Asp Arg Asp Cys Ala Ala Leu
Ala Ser Asn Gly 180 185 190 Glu Tyr Ser Ile Ala Asn Asn Gly Val Ala
Asn Tyr Lys Ala Tyr Ile 195 200 205 Asp Ala Ile Arg Ala Gln Leu Val
Lys Tyr Ser Asp Val His Thr Ile 210 215 220 Leu Val Ile Glu Pro Asp
Ser Leu Ala Asn Leu Val Thr Asn Leu Asn 225 230 235 240 Val Ala Lys
Cys Ala Asn Ala Gln Ser Ala Tyr Leu Glu Cys Val Asp 245 250 255 Tyr
Ala Leu Lys Gln Leu Asn Leu Pro Asn Val Ala Met Tyr Leu Asp 260 265
270 Ala Gly His Ala Gly Trp Leu Gly Trp Pro Ala Asn Leu Gly Pro Ala
275 280 285 Ala Thr Leu Phe Ala Lys Val Tyr Thr Asp Ala Gly Ser Pro
Ala Ala 290 295 300 Val Arg Gly Leu Ala Thr Asn Val Ala Asn Tyr Asn
Ala Trp Ser Leu 305 310 315 320 Ser Thr Cys Pro Ser Tyr Thr Gln Gly
Asp Pro Asn Cys Asp Glu Lys 325 330 335 Lys Tyr Ile Asn Ala Met Ala
Pro Leu Leu Lys Glu Ala Gly Phe Asp 340 345 350 Ala His Phe Ile Met
Asp Thr Ser Arg Asn Gly Val Gln Pro Thr Lys 355 360 365 Gln Asn Ala
Trp Gly Asp Trp Cys Asn Val Ile Gly Thr Gly Phe Gly 370 375 380 Val
Arg Pro Ser Thr Asn Thr Gly Asp Pro Leu Gln Asp Ala Phe Val 385 390
395 400 Trp Ile Lys Pro Gly Gly Glu Ser Asp Gly Thr Ser Asn Ser Thr
Ser 405 410 415 Pro Arg Tyr Asp Ala His Cys Gly Tyr Ser Asp Ala Leu
Gln Pro Ala 420 425 430 Pro Glu Ala Gly Thr Trp Phe Gln Ala Tyr Phe
Glu Gln Leu Leu Thr 435 440 445 Asn Ala Asn Pro Ser Phe 450
8344PRTAspergillus aculeatusSIGNAL(1)..(22)mat_peptide(23)..(344)
8Met Val Gly Leu Leu Ser Ile Thr Ala Ala Leu Ala Ala Thr Val Leu
-20 -15 -10 Pro Asn Ile Val Ser Ala Val Gly Leu Asp Gln Ala Ala Val
Ala Lys -5 -1 1 5 10 Gly Leu Gln Tyr Phe Gly Thr Ala Thr Asp Asn
Pro Glu Leu Thr Asp 15 20 25 Ile Pro Tyr Val Thr Gln Leu Asn Asn
Thr Ala Asp Phe Gly Gln Ile 30 35 40 Thr Pro Gly Asn Ser Met Lys
Trp Asp Ala Thr Glu Pro Ser Gln Gly 45 50 55 Thr Phe Thr Phe Thr
Lys Gly Asp Val Ile Ala Asp Leu Ala Glu Gly 60 65 70 Asn Gly Gln
Tyr Leu Arg Cys His Thr Leu Val Trp Tyr Asn Gln Leu 75 80 85 90 Pro
Ser Trp Val Thr Ser Gly Thr Trp Thr Asn Ala Thr Leu Thr Ala 95 100
105 Ala Leu Lys Asn His Ile Thr Asn Val Val Ser His Tyr Lys Gly Lys
110 115 120 Cys Leu His Trp Asp Val Val Asn Glu Ala Leu Asn Asp Asp
Gly Thr 125 130 135 Tyr Arg Thr Asn Ile Phe Tyr Thr Thr Ile Gly Glu
Ala Tyr Ile Pro 140 145 150 Ile Ala Phe Ala Ala Ala Ala Ala Ala Asp
Pro Asp Ala Lys Leu Phe 155 160 165 170 Tyr Asn Asp Tyr Asn Leu Glu
Tyr Gly Gly Ala Lys Ala Ala Ser Ala 175 180 185 Arg Ala Ile Val Gln
Leu Val Lys Asn Ala Gly Ala Lys Ile Asp Gly 190 195 200 Val Gly Leu
Gln Ala His Phe Ser Val Gly Thr Val Pro Ser Thr Ser 205 210 215 Ser
Leu Val Ser Val Leu Gln Ser Phe Thr Ala Leu Gly Val Glu Val 220 225
230 Ala Tyr Thr Glu Ala Asp Val Arg Ile Leu Leu Pro Thr Thr Ala Thr
235 240 245 250 Thr Leu Ala Gln Gln Ser Ser Asp Phe Gln Ala Leu Val
Gln Ser Cys 255 260 265 Val Gln Thr Thr Gly Cys Val Gly Phe Thr Ile
Trp Asp Trp Thr Asp 270 275 280 Lys Tyr Ser Trp Val Pro Ser Thr Phe
Ser Gly Tyr Gly Ala Ala Leu 285 290 295 Pro Trp Asp Glu Asn Leu Val
Lys Lys Pro Ala Tyr Asn Gly Leu Leu 300 305 310 Ala Gly Met Gly Val
Thr Val Thr 315 320 9397PRTAspergillus fumigatus 9Met Val His Leu
Ser Ser Leu Ala Ala Ala Leu Ala Ala Leu Pro Leu 1 5 10 15 Val Tyr
Gly Ala Gly Leu Asn Thr Ala Ala Lys Ala Lys Gly Leu Lys 20 25 30
Tyr Phe Gly Ser Ala Thr Asp Asn Pro Glu Leu Thr Asp Ser Ala Tyr 35
40 45 Val Ala Gln Leu Ser Asn Thr Asp Asp Phe Gly Gln Ile Thr Pro
Gly 50 55 60 Asn Ser Met Lys Trp Asp Ala Thr Glu Pro Ser Gln Asn
Ser Phe Ser 65 70 75 80 Phe Ala Asn Gly Asp Ala Val Val Asn Leu Ala
Asn Lys Asn Gly Gln 85 90 95 Leu Met Arg Cys His Thr Leu Val Trp
His Ser Gln Leu Pro Asn Trp 100 105 110 Val Ser Ser Gly Ser Trp Thr
Asn Ala Thr Leu Leu Ala Ala Met Lys 115 120 125 Asn His Ile Thr Asn
Val Val Thr His Tyr Lys Gly Lys Cys Tyr Ala 130 135 140 Trp Asp Val
Val Asn Glu Ala Leu Asn Glu Asp Gly Thr Phe Arg Asn 145 150 155 160
Ser Val Phe Tyr Gln Ile Ile Gly Pro Ala Tyr Ile Pro Ile Ala Phe 165
170 175 Ala Thr Ala Ala Ala Ala Asp Pro Asp Val Lys Leu Tyr Tyr Asn
Asp 180 185 190 Tyr Asn Ile Glu Tyr Ser Gly Ala Lys Ala Thr Ala Ala
Gln Asn Ile 195 200 205 Val Lys Met Ile Lys Ala Tyr Gly Ala Lys Ile
Asp Gly Val Gly Leu 210 215 220 Gln Ala His Phe Ile Val Gly Ser Thr
Pro Ser Gln Ser Asp Leu Thr 225 230 235 240 Thr Val Leu Lys Gly Tyr
Thr Ala Leu Gly Val Glu Val Ala Tyr Thr 245 250 255 Glu Leu Asp Ile
Arg Met Gln Leu Pro Ser Thr Ala Ala Lys Leu Ala 260 265 270 Gln Gln
Ser Thr Asp Phe Gln Gly Val Ala Ala Ala Cys Val Ser Thr 275 280 285
Thr Gly Cys Val Gly Val Thr Ile Trp Asp Trp Thr Asp Lys Tyr Ser 290
295 300 Trp Val Pro Ser Val Phe Gln Gly Tyr Gly Ala Pro Leu Pro Trp
Asp 305 310 315 320 Glu Asn Tyr Val Lys Lys Pro Ala Tyr Asp Gly Leu
Met Ala Gly Leu 325 330 335 Gly Ala Ser Gly Ser Gly Thr Thr Thr Thr
Thr Thr Thr Thr Ser Thr 340 345 350 Thr Thr Gly Gly Thr Asp Pro Thr
Gly Val Ala Gln Lys Trp Gly Gln 355 360 365 Cys Gly Gly Ile Gly Trp
Thr Gly Pro Thr Thr Cys Val Ser Gly Thr 370 375 380 Thr Cys Gln Lys
Leu Asn Asp Trp Tyr Ser Gln Cys Leu 385 390 395 10792PRTAspergillus
fumigatus 10Met Ala Val Ala Lys Ser Ile Ala Ala Val Leu Val Ala Leu
Leu Pro 1 5 10 15 Gly Ala Leu Ala Gln Ala Asn Thr Ser Tyr Val Asp
Tyr Asn Val Glu 20 25 30 Ala Asn Pro Asp Leu Thr Pro Gln Ser Val
Ala Thr Ile Asp Leu Ser 35 40 45 Phe Pro Asp Cys Glu Asn Gly Pro
Leu Ser Lys Thr Leu Val Cys Asp 50 55 60 Thr Ser Ala Arg Pro His
Asp Arg Ala Ala Ala Leu Val Ser Met Phe 65 70 75 80 Thr Phe Glu Glu
Leu Val Asn Asn Thr Gly Asn Thr Ser Pro Gly Val 85 90 95 Pro Arg
Leu Gly Leu Pro Pro Tyr Gln Val Trp Ser Glu Ala Leu His 100 105 110
Gly Leu Asp Arg Ala Asn Phe Thr Asn Glu Gly Glu Tyr Ser Trp Ala 115
120 125 Thr Ser Phe Pro Met Pro Ile Leu Thr Met Ser Ala Leu Asn Arg
Thr 130 135 140 Leu Ile Asn Gln Ile Ala Thr Ile Ile Ala Thr Gln Gly
Arg Ala Phe 145 150 155 160 Asn Asn Val Gly Arg Tyr Gly Leu Asp Val
Tyr Ala Pro Asn Ile Asn 165 170 175 Ala Phe Arg Ser Ala Met Trp Gly
Arg Gly Gln Glu Thr Pro Gly Glu 180 185 190 Asp Ala Tyr Cys Leu Ala
Ser Ala Tyr Ala Tyr Glu Tyr Ile Thr Gly 195 200 205 Ile Gln Gly Gly
Val Asp Pro Glu His Leu Lys Leu Val Ala Thr Ala 210 215 220 Lys His
Tyr Ala Gly Tyr Asp Leu Glu Asn Trp Asp Gly His Ser Arg 225 230 235
240 Leu Gly Asn Asp Met Asn Ile Thr Gln Gln Glu Leu Ser Glu Tyr Tyr
245 250 255 Thr Pro Gln Phe Leu Val Ala Ala Arg Asp Ala Lys Val His
Ser Val 260 265 270 Met Cys Ser Tyr Asn Ala Val Asn Gly Val Pro Ser
Cys Ala Asn Ser 275 280 285 Phe Phe Leu Gln Thr Leu Leu Arg Asp Thr
Phe Gly Phe Val Glu Asp 290 295 300 Gly Tyr Val Ser Ser Asp Cys Asp
Ser Ala Tyr Asn Val Trp Asn Pro 305 310 315 320 His Glu Phe Ala Ala
Asn Ile Thr Gly Ala Ala Ala Asp Ser Ile Arg 325 330 335 Ala Gly Thr
Asp Ile Asp Cys Gly Thr Thr Tyr Gln Tyr Tyr Phe Gly 340 345 350 Glu
Ala Phe Asp Glu Gln Glu Val Thr Arg Ala Glu Ile Glu Arg Gly 355 360
365 Val Ile Arg Leu Tyr Ser Asn Leu Val Arg Leu Gly Tyr Phe Asp Gly
370 375 380 Asn Gly Ser Val Tyr Arg Asp Leu Thr Trp Asn Asp Val Val
Thr Thr 385 390 395 400 Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala Val
Glu Gly Ile Val Leu 405 410 415 Leu Lys Asn Asp Gly Thr Leu Pro Leu
Ala Lys Ser Val Arg Ser Val 420 425 430 Ala Leu Ile Gly Pro Trp Met
Asn Val Thr Thr Gln Leu Gln Gly Asn 435 440 445 Tyr Phe Gly Pro Ala
Pro Tyr Leu Ile Ser Pro Leu Asn Ala Phe Gln 450 455 460 Asn Ser Asp
Phe Asp Val Asn Tyr Ala Phe Gly Thr Asn Ile Ser Ser 465 470 475 480
His Ser Thr Asp Gly Phe Ser Glu Ala Leu Ser Ala Ala Lys Lys Ser 485
490 495 Asp Val Ile Ile Phe Ala Gly Gly Ile Asp Asn Thr Leu Glu Ala
Glu 500 505 510 Ala Met Asp Arg Met Asn Ile Thr Trp Pro Gly Asn Gln
Leu Gln Leu 515 520 525 Ile Asp Gln Leu Ser Gln Leu Gly Lys Pro Leu
Ile Val Leu Gln Met 530 535 540 Gly Gly Gly Gln Val Asp Ser Ser Ser
Leu Lys Ser Asn Lys Asn Val 545 550 555 560 Asn Ser Leu Ile Trp Gly
Gly Tyr Pro Gly Gln Ser Gly Gly Gln Ala 565 570 575 Leu Leu Asp Ile
Ile Thr Gly Lys Arg Ala Pro Ala Gly Arg Leu Val 580 585 590 Val Thr
Gln Tyr Pro Ala Glu Tyr Ala Thr Gln Phe Pro Ala Thr Asp 595 600 605
Met Ser Leu Arg Pro His Gly Asn Asn Pro Gly Gln Thr Tyr Met Trp 610
615 620 Tyr Thr Gly Thr Pro Val Tyr Glu Phe Gly His Gly Leu Phe Tyr
Thr 625 630 635 640 Thr Phe His Ala Ser Leu Pro Gly Thr Gly Lys Asp
Lys Thr Ser Phe 645 650 655 Asn Ile Gln Asp Leu Leu Thr Gln Pro His
Pro Gly Phe Ala Asn Val 660 665 670 Glu Gln Met Pro Leu Leu Asn Phe
Thr Val Thr Ile Thr Asn Thr Gly 675 680 685 Lys Val Ala Ser Asp Tyr
Thr Ala Met Leu Phe Ala Asn
Thr Thr Ala 690 695 700 Gly Pro Ala Pro Tyr Pro Asn Lys Trp Leu Val
Gly Phe Asp Arg Leu 705 710 715 720 Ala Ser Leu Glu Pro His Arg Ser
Gln Thr Met Thr Ile Pro Val Thr 725 730 735 Ile Asp Ser Val Ala Arg
Thr Asp Glu Ala Gly Asn Arg Val Leu Tyr 740 745 750 Pro Gly Lys Tyr
Glu Leu Ala Leu Asn Asn Glu Arg Ser Val Val Leu 755 760 765 Gln Phe
Val Leu Thr Gly Arg Glu Ala Val Ile Phe Lys Trp Pro Val 770 775 780
Glu Gln Gln Gln Ile Ser Ser Ala 785 790 11797PRTTrichoderma reesei
11Met Val Asn Asn Ala Ala Leu Leu Ala Ala Leu Ser Ala Leu Leu Pro 1
5 10 15 Thr Ala Leu Ala Gln Asn Asn Gln Thr Tyr Ala Asn Tyr Ser Ala
Gln 20 25 30 Gly Gln Pro Asp Leu Tyr Pro Glu Thr Leu Ala Thr Leu
Thr Leu Ser 35 40 45 Phe Pro Asp Cys Glu His Gly Pro Leu Lys Asn
Asn Leu Val Cys Asp 50 55 60 Ser Ser Ala Gly Tyr Val Glu Arg Ala
Gln Ala Leu Ile Ser Leu Phe 65 70 75 80 Thr Leu Glu Glu Leu Ile Leu
Asn Thr Gln Asn Ser Gly Pro Gly Val 85 90 95 Pro Arg Leu Gly Leu
Pro Asn Tyr Gln Val Trp Asn Glu Ala Leu His 100 105 110 Gly Leu Asp
Arg Ala Asn Phe Ala Thr Lys Gly Gly Gln Phe Glu Trp 115 120 125 Ala
Thr Ser Phe Pro Met Pro Ile Leu Thr Thr Ala Ala Leu Asn Arg 130 135
140 Thr Leu Ile His Gln Ile Ala Asp Ile Ile Ser Thr Gln Ala Arg Ala
145 150 155 160 Phe Ser Asn Ser Gly Arg Tyr Gly Leu Asp Val Tyr Ala
Pro Asn Val 165 170 175 Asn Gly Phe Arg Ser Pro Leu Trp Gly Arg Gly
Gln Glu Thr Pro Gly 180 185 190 Glu Asp Ala Phe Phe Leu Ser Ser Ala
Tyr Thr Tyr Glu Tyr Ile Thr 195 200 205 Gly Ile Gln Gly Gly Val Asp
Pro Glu His Leu Lys Val Ala Ala Thr 210 215 220 Val Lys His Phe Ala
Gly Tyr Asp Leu Glu Asn Trp Asn Asn Gln Ser 225 230 235 240 Arg Leu
Gly Phe Asp Ala Ile Ile Thr Gln Gln Asp Leu Ser Glu Tyr 245 250 255
Tyr Thr Pro Gln Phe Leu Ala Ala Ala Arg Tyr Ala Lys Ser Arg Ser 260
265 270 Leu Met Cys Ala Tyr Asn Ser Val Asn Gly Val Pro Ser Cys Ala
Asn 275 280 285 Ser Phe Phe Leu Gln Thr Leu Leu Arg Glu Ser Trp Gly
Phe Pro Glu 290 295 300 Trp Gly Tyr Val Ser Ser Asp Cys Asp Ala Val
Tyr Asn Val Phe Asn 305 310 315 320 Pro His Asp Tyr Ala Ser Asn Gln
Ser Ser Ala Ala Ala Ser Ser Leu 325 330 335 Arg Ala Gly Thr Asp Ile
Asp Cys Gly Gln Thr Tyr Pro Trp His Leu 340 345 350 Asn Glu Ser Phe
Val Ala Gly Glu Val Ser Arg Gly Glu Ile Glu Arg 355 360 365 Ser Val
Thr Arg Leu Tyr Ala Asn Leu Val Arg Leu Gly Tyr Phe Asp 370 375 380
Lys Lys Asn Gln Tyr Arg Ser Leu Gly Trp Lys Asp Val Val Lys Thr 385
390 395 400 Asp Ala Trp Asn Ile Ser Tyr Glu Ala Ala Val Glu Gly Ile
Val Leu 405 410 415 Leu Lys Asn Asp Gly Thr Leu Pro Leu Ser Lys Lys
Val Arg Ser Ile 420 425 430 Ala Leu Ile Gly Pro Trp Ala Asn Ala Thr
Thr Gln Met Gln Gly Asn 435 440 445 Tyr Tyr Gly Pro Ala Pro Tyr Leu
Ile Ser Pro Leu Glu Ala Ala Lys 450 455 460 Lys Ala Gly Tyr His Val
Asn Phe Glu Leu Gly Thr Glu Ile Ala Gly 465 470 475 480 Asn Ser Thr
Thr Gly Phe Ala Lys Ala Ile Ala Ala Ala Lys Lys Ser 485 490 495 Asp
Ala Ile Ile Tyr Leu Gly Gly Ile Asp Asn Thr Ile Glu Gln Glu 500 505
510 Gly Ala Asp Arg Thr Asp Ile Ala Trp Pro Gly Asn Gln Leu Asp Leu
515 520 525 Ile Lys Gln Leu Ser Glu Val Gly Lys Pro Leu Val Val Leu
Gln Met 530 535 540 Gly Gly Gly Gln Val Asp Ser Ser Ser Leu Lys Ser
Asn Lys Lys Val 545 550 555 560 Asn Ser Leu Val Trp Gly Gly Tyr Pro
Gly Gln Ser Gly Gly Val Ala 565 570 575 Leu Phe Asp Ile Leu Ser Gly
Lys Arg Ala Pro Ala Gly Arg Leu Val 580 585 590 Thr Thr Gln Tyr Pro
Ala Glu Tyr Val His Gln Phe Pro Gln Asn Asp 595 600 605 Met Asn Leu
Arg Pro Asp Gly Lys Ser Asn Pro Gly Gln Thr Tyr Ile 610 615 620 Trp
Tyr Thr Gly Lys Pro Val Tyr Glu Phe Gly Ser Gly Leu Phe Tyr 625 630
635 640 Thr Thr Phe Lys Glu Thr Leu Ala Ser His Pro Lys Ser Leu Lys
Phe 645 650 655 Asn Thr Ser Ser Ile Leu Ser Ala Pro His Pro Gly Tyr
Thr Tyr Ser 660 665 670 Glu Gln Ile Pro Val Phe Thr Phe Glu Ala Asn
Ile Lys Asn Ser Gly 675 680 685 Lys Thr Glu Ser Pro Tyr Thr Ala Met
Leu Phe Val Arg Thr Ser Asn 690 695 700 Ala Gly Pro Ala Pro Tyr Pro
Asn Lys Trp Leu Val Gly Phe Asp Arg 705 710 715 720 Leu Ala Asp Ile
Lys Pro Gly His Ser Ser Lys Leu Ser Ile Pro Ile 725 730 735 Pro Val
Ser Ala Leu Ala Arg Val Asp Ser His Gly Asn Arg Ile Val 740 745 750
Tyr Pro Gly Lys Tyr Glu Leu Ala Leu Asn Thr Asp Glu Ser Val Lys 755
760 765 Leu Glu Phe Glu Leu Val Gly Glu Glu Val Thr Ile Glu Asn Trp
Pro 770 775 780 Leu Glu Glu Gln Gln Ile Lys Asp Ala Thr Pro Asp Ala
785 790 795
* * * * *